Advances in mass spectrometry imaging for toxicological analysis and safety evaluation of pharmaceuticals.

Safety issues caused by pharmaceuticals have frequently occurred worldwide, posing a tremendous threat to human health. As an essential part of drug development, the toxicological analysis and safety evaluation is of great significance. In addition, the risk of pharmaceuticals accumulation in the environment and the monitoring of the toxicity from natural medicines have also received ongoing concerns. Due to a lack of spatial distribution information provided by common analytical methods, analyses that provide spatial dimensions could serve as complementary safety evaluation methods for better prediction and evaluation of drug toxicity. With advances in technical solutions and software algorithms, mass spectrometry imaging (MSI) has received increasing attention as a popular analytical tool that enables the simultaneous implementation of qualitative, quantitative, and localization without complex sample pretreatment and labeling steps. In recent years, MSI has become more attractive, powerful, and sensitive and has been applied in several scientific fields that can meet the safety assessment requirements. This review aims to cover a detailed summary of the various MSI technologies utilized in the biomedical and pharmaceutical area, including technical principles, advantages, current status, and future trends. Representative applications and developments in the safety-related issues of different pharmaceuticals and natural medicines are also described to provide a reference for pharmaceutical research, improve rational clinical medicine use, and ensure public safety.

On-site analysis and rapid identification of citrus herbs by miniature mass spectrometry and machine learning.

BACKGROUND: Natural medicines present a considerable analytical challenge due to their diverse botanical origins and complex multi-species composition. This inherent complexity complicates their rapid identification and analysis. Tangerine peel, a product of the Citrus species from the Rutaceae family, is widely used both as a culinary ingredient and in traditional Chinese medicine. It is classified into two primary types in China: Citri Reticulatae Pericarpium (CP) and Citri Reticulatae Pericarpium Viride (QP), differentiated by harvest time. A notable price disparity exists between CP and another variety, Citri reticulatae "Chachi" (GCP), with differences being based on the original variety. METHODS: This study introduces an innovative method using portable miniature mass spectrometry for swift on-site analysis of QP, CP, and GCP, requiring less than a minute per sample. And combined with machine learning to differentiate the three types on site, the method was used to try to distinguish GCP from different storage years. RESULTS: This novel method using portable miniature mass spectrometry for swift on-site analysis of tangerine peels enabled the characterization of 22 compounds in less than one minute per sample. The method simplifies sample processing and integrates machine learning to distinguish between the CP, QP, and GCP varieties. Moreover, a multiple-perceptron neural network model is further employed to specifically differentiate between CP and GCP, addressing the significant price gap between them. CONCLUSIONS: The entire analytical time of the method is about 1 minute, and samples can be analyzed on site, greatly reducing the cost of testing. Besides, this approach is versatile, operates independently of location and environmental conditions, and offers a valuable tool for assessing the quality of natural medicines.

Rapid analysis of bioactive compounds from citrus samples by direct analysis in real-time mass spectrometry combined with chemometrics.

Renowned for their nutritional benefits, citrus fruits are harvested at various stages in China for functional food production. This study introduces an innovative analytical method, DART-MS, enabling direct qualitative analysis of citrus samples without the need for preprocessing. Simultaneously, the combination of chemometrics can be applied to distinguish between three different citrus samples: Citri Reticulatae Pericarpium, Citri Reticulatae Pericarpium Viride, and Citri Reticulatae "Chachi". Notably, given the international regulatory concerns surrounding synephrine, a precise quantitative analysis method for synephrine was developed. The limit of detection (LOD) and the limit of quantification (LOQ) were 39 ng mL-1 and 156 ng mL-1, respectively. The recovery rates obtained varied from 98.46% to 100.71%. Furthermore, the intra-day and inter-day precision demonstrated robust consistency, with values spanning 5.0-6.1% and 5.03-6.08%, respectively, offering quicker results compared to those from HPLC-MS, promising a safer assessment of herbal and food products.

[Natural 5α-reductase inhibitors in treatment of benign prostatic hyperplasia].

Benign prostatic hyperplasia(BPH) is a common disease of the male urinary system, and its incidence rate in China is increasing. However, the mechanism underlying the pathogenesis of BPH remains unclear. Some studies demonstrated that the incidence of BPH was related to the change in the levels of steroid hormones. Too high content of dihydrotestosterone(DHT) in the body may cause BPH and other related diseases. Testosterone(T) is converted to DHT by 5α-reductase(SRD5A). By inhibiting the activity of this enzyme, the production of DHT can be reduced, and then the incidence of BPH can be lowered. Therefore, it has drawn great attention to screen and discover safer and more effective 5α-reductase inhibitors from natural medicines to treat prostatic hyperplasia without affecting the physiological function of men. This review summarizes the characteristics and tissue distribution of 5α-reductase, the discovery of 5α-reductase inhibitors in traditional Chinese medicine and natural medicines, 5α-reductase inhibitors commonly used in clinical practice and their side effects, as well as the animal models of prostatic hyperplasia and common detection indicators, aiming to provide a reference for more in-depth understanding and research about BPH and development of drugs.

A comprehensive profiling of renin-angiotensin system in mouse and human plasma by a rapid quantitative analysis of 14 angiotensin peptides using ultrahigh-performance liquid chromatography with tandem mass spectrometry.

BACKGROUND: The renin-angiotensin system produces a series of biologically active angiotensin (Ang) peptides. These Ang peptides are the major regulators of blood pressure and Na homeostasis, and play a critical role in maintaining cardiovascular and fluid homeostasis. The concentration of Ang peptides in the body is at trace levels, making their detection and quantification a challenge. In this study, a rapid and sensitive analytical method using mass spectrometry coupled with ultrahigh-performance liquid chromatography (UHPLC/MS) was developed to simultaneously quantify 14 Ang peptides. METHODS: UHPLC/MS was employed to quantify 14 Ang peptides in mouse and human plasma. An HSS T3 column (2.1 × 100 mm, 1.8 µm) with an HSS T3 precolumn and triple-quadrupole mass spectrometer combined with an electrospray ionization source were utilized. Sample pretreatment involved a one-step protein precipitation using methanol. The total analysis time was within 7.5 min and the target peptides were detected in positive ion mode and quantified by selected reaction monitoring mode. RESULTS: The method was validated for linearity, detection and quantification limits, precision, stability, recovery and matrix effect. The limits of detection of Ang II, Ang III, Ang-(1-7), Ang-(2-7), Ang-(3-7), Ang-(1-9), bradykinin, Asn1 and Val5 -Ang II are all less than 1 pg mL-1 , indicating high sensitivity. The intra-day and inter-day precision was within 15%, and the accuracy was between 85% and 115%. Meanwhile, the sample and reference solution were stable within 48 h, and the recovery and matrix effect met the quantitative requirements. CONCLUSIONS: The method is currently reported to allow the largest number of Ang peptide species to be detected at one time. In addition, the proposed method offers a fast and reliable approach for comprehensive analysis of Ang metabolism in biological samples, facilitating research on the physiological and pathological states of cardiovascular, kidney and respiratory diseases.

Rapid identification of bear bile powder from other bile sources using chip-based nano-electrospray ionization tandem mass spectrometry.

RATIONALE: Bear bile powder (BBP) is a widely used traditional Chinese medicine (TCM), and bile acids (BAs) are the main active components in BBP. Due to the scarcity of BBP resources, adulterations often occur in the market. Conventional methods to distinguish them are usually complicated and time-consuming. To enhance effectiveness and accuracy, a rapid and rough analytical method is desperately needed. METHODS: In this study, a rapid strategy using chip-based nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was established to distinguish BBP from other sources of bile powder (BP). In addition, the results were further verified by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UPLC/MS). RESULTS: The precision of the chip-based nano-ESI-MS/MS method was validated to be acceptable with relative standard deviation (RSD) <15%. The distinction between BBP and other sources of BP, including common adulterants of pig bile powder (PBP), cattle bile powder (CBP), sheep bile powder (SBP), and chicken bile powder (CkBP), can be observed in the spectra. By using orthogonal partial least-squares discriminant analysis (OPLS-DA), more potential m/z markers were investigated. A BAs-related m/z marker of 498.3 was discovered as a typical differential molecular ion peak and was identified as tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) in BBP. CONCLUSIONS: The proposed strategy has simple sample pretreatment steps and significantly shortened analysis time. As an emerging technology, chip-based nano-ESI-MS not only provides a reference for the rapid distinction of adulterated Chinese medicines, but also provides some insights into the identification of other chemicals and foods.

Rapid analysis of differential chemical compositions of Poria cocos using thin-layer chromatography spray ionization-mass spectrometry.

Poria cocos, with medicinal and edible properties, contains multiple chemical components that pose difficulties in its quality control. Herein, we established a simple thin-layer chromatography spray ionization-mass spectrometry (TLCSI-MS) device that allows the simultaneous separation and identification of bioactive compounds. The triterpene acids in different medicinal parts of Poria cocos were characterized, and the markers of differences were revealed. The quantitative analysis of its pharmaceutical preparation has also been performed to verify the feasibility. The study provides a new analytical perspective for the screening and identification of multiple compounds and a good option for the quality evaluation of herbal medicines and foods.

Development of Green and Efficient Extraction of Bioactive Ginsenosides from Panax ginseng with Deep Eutectic Solvents.

The extraction of active constituents from natural sources in a green and efficient manner is considered an important field in the pharmaceutical industry. In recent years, deep eutectic solvents (DESs), a new type of green solvent, have attracted increasing attention. Therefore, we aimed to establish a green and high-efficiency extraction method for ginsenosides based on DESs. This study takes Panax ginseng as a model sample. Eighteen different DESs were produced to extract polar ginsenosides. Ultrasound-assisted extraction (UAE) was applied for simplicity and efficiency. A binary DES synthesized using choline chloride and urea at a proportion of 1:2 prepared by a heating stirring method is proven to be more effective than other solvents, such as the widely used 70% ethanol for the extraction of ginsenosides. Three variables that might affect the extraction, including the DES content in the extraction solvent, liquid/solid ratio, and ultrasound extraction time, were evaluated for optimization. The optimum extraction conditions for ginsenosides were determined as follows: DES water content of 20 wt%, liquid/solid ratio of 15 mL g-1, and an ultrasonic extraction time of 15 min. The extraction yield for the optimized method is found to be 31% higher than that for 70% ethanol, which achieves efficient extraction. This study shows that DESs are available to extract ginsenosides for use in traditional Chinese medicine. The discovery also contributes to further research into the green extraction of ginsenosides.

[Research advances of chemical constituents and analytical methods of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium].

Citri Reticulatae Pericarpium Viride(CRPV) and Citri Reticulatae Pericarpium(CRP) are two commonly used Chinese medicinal materials. They have the same origin while are harvested in different seasons and have different clinical effects. They contain similar chemical components, like flavonoids, terpenes, volatile oils, and alkaloids. Although it has been demonstrated that differential components exist between them, there is still a lack of systematic comparison. Many studies have reported the chemical composition and quality evaluation of CRPV and CRP, including the characterization of flavonoids, alkaloids, and volatile oils via thin-layer chromatography, high-performance liquid chromatography, gas chromatography, infrared spectroscopy, and ultraviolet spectroscopy. A few studies have explored the differences between CRPV and CRP. In this paper, we systematically summarized the reported chemical composition, analytical methods, and quality evaluation of CRPV and CRP in recent ten years, aiming to facilitate the research on the pharmacodynamic material basis, quality evaluation, and standard improvement of CRPV and CRP.

[New method for green extraction of ginsenosides based on mechanochemically-assisted extraction and deep eutectic solvents].

Ginsenosides are the main active ingredients in ginseng. Studies have shown that ginsenosides have anti-virus, anti-tumor, anti-aging, nootropic, cardiovascular diseases-protecting, and other pharmacological activities. Thus, the development and utilization of ginsenosides have persistently attracted much attention. At present, the extraction of ginsenosides is mainly based on organic solvents, and there are relatively few studies on their green extraction. In this study, different deep eutectic solvents(DESs) were synthesized by heating and stirring method, combined with an emerging technology, mechanochemically-assisted extraction(MCAE), to extract ginsenosides in a green way. Six parameters that might affect the extraction effect were optimized to determine the optimal conditions, and the method validation was conducted. The new established method was compared with a commonly used extraction method(ultrasound-assisted extraction using 70% ethanol) to evaluate its extraction efficiency. The results revealed that the optimal extraction conditions of DES-MCAE were that the volume ratio of DES3(choline chloride∶urea 1∶2) and water in the extraction solvent was 6∶4, and the liquid ratio and the linear vibration speed were 0.05 g·mL~(-1) and 4.0 m·s~(-1); the extraction was performed twice, 40 s each. With only 80 s extraction, the extraction rate of this method was 36.22% higher than that of ultrasoun-dassisted extraction using 70% ethanol. In this study, a DESs-based pretreatment method for ginsenosides was established and its rapid, green and efficient extraction was realized, which provided new ideas and methods for further research on green extraction of other active ingredients from Chinese medicine.

Development and applications of deep eutectic solvent derived functional materials in chromatographic separation.

Deep eutectic solvents have received increasing attention over the past decade, and the green and versatile nature makes them important media for developing environmentally friendly and sustainable technologies. In addition to act as green alternatives to traditional organic solvents, deep eutectic solvents offer tremendous opportunities to produce different kinds of emerging functional materials. The present review highlights the recent development and applications of deep functional materials based on deep eutectic solvents as novel adsorbents for diverse analytes in complex matrices. The type, preparation, and unique properties of deep eutectic solvents, the synthesis of deep eutectic solvent derived materials, as well as their applications in chromatographic separation are described. We aim to demonstrate that deep eutectic solvents not only allow the design of eco-friendly extraction processes but also open straightforward access to advanced materials in sample preparation.

Fully automated chip-based nanoelectrospray ionization-mass spectrometry as an effective tool for rapid and high-throughput screening of 5α-reductase inhibitors.

The 5α-reductase converts testosterone to dihydrotestosterone (DHT), and excess DHT could cause androgen-related diseases such as androgenetic alopecia and benign prostatic hyperplasia (BPH). To discover new 5α-reductase inhibitors, effective drug screening method with high throughput is thus essential. In this study, fully automated chip-based nanoelectrospray ionization-mass spectrometry (nano-ESI-MS) was innovatively utilized as a screening tool for 5α-reductase inhibitory assay in direct infusion mode, which simplified sample pretreatment and greatly improved experimental efficiency. The preliminary data indicated that curcumin, a natural anti-inflammatory compound, exhibited notably 5α-reductase inhibition activity. Moreover, the obtained results of the chip-based nano-ESI-MS were well consistent with those of HPLC-MS, which suggested that the chip-based nano-ESI-MS could be treated as a rapid and high-throughput drugs screening strategy in pharmaceutical development. Graphical abstract.

Rapid and sensitive analysis of progesterone by solid-phase extraction with amino-functionalized metal-organic frameworks coupled to direct analysis in real-time mass spectrometry.

Progesterone is the representative progestogens in five major classes of steroid hormones and plays important roles in mammalian pregnancy and animal growth and development. Conventional available analytical methods for progesterone involve immunoassay, gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography-mass spectrometry (HPLC-MS), which lack specificity or usually require sophisticated operations and relatively long time. Herein, we developed a novel strategy for rapid analysis of progesterone via direct analysis in real time mass spectrometry (DART-MS) combined with solid-phase extraction (SPE) using an amino functionalized metal-organic frameworks (MOFs). Under optimized conditions, a wide linear range of 0.5-500 ng mL-1 was achieved, with a satisfactory correlation coefficient (R2 = 0.9992). The relative standard deviations (RSDs) were in the range from 2.4 to 8.4%, demonstrating good precision. The applicability was then confirmed by analyzing spiked lake water and synthetic urine samples, and recoveries are between 92.0 and 117.8% in all three spiked levels (5, 25, and 100 ng mL-1). The sensitivity was notably improved compared with solely DART-MS and obtained detection limit decreased by about 50 times. This research provided a rapid, simple, highly sensitive, and efficient approach for analysis of hormones through combination of advantages of ambient mass spectrometry and porous nanomaterials. Graphical abstract.

Rapid authentication of agarwood by using liquid extraction surface analysis mass spectrometry (LESA-MS).

INTRODUCTION: Agarwood is a highly valuable fragrant resinous wood which is widely used as traditional Chinese medicines, perfumes, incense and decorations. Due to its high economic value and excessive demand, this leads to a rising price and proliferation of fake commodities. Thus, strict authenticity identification and quality evaluation of agarwood are of great significance. OBJECTIVE: To establish a simple, rapid and non-destructive technique for identifying the authenticity of agarwood. METHODS: Liquid extraction surface analysis mass spectrometry (LESA-MS) was firstly proposed to identify the authenticity of 62 agarwood samples without sample preparation. In addition, multivariate statistical models and thin-layer chromatography (TLC) method were used to analyse and verify the results of LESA-MS. RESULTS: Representative compounds of agarwood were detected by LESA-MS. A characteristic 2-(2-phenylethyl)chromone compound (m/z 319.1) was treated as a key chemical marker to identify agarwood and its counterfeits rapidly. Several other chromones ions were identified and used as additional evidence for authentic samples. A total of 62 samples were visually discriminated as two groups by principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA), and the specific characteristic marker was highlighted. Moreover, the qualitative results of the conventional TLC method were in agreement with the LESA-MS approach. CONCLUSION: The proposed LESA-MS method was successfully applied in the direct qualitative analysis of agarwood from different sources. This study indicated great feasibility and practicality of LESA-MS in the rapid identification of agarwood, and provided a non-destructive and meaningful preliminary screening tool for the agarwood industry.

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin ß1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinß1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKß induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1ß), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinß1-siRNA or the JNK inhibitor. We also found that integrinß1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

Facile one-step solvothermal synthesis of a luminescent europium metal-organic framework for rapid and selective sensing of uranyl ions.

A simple, cost-effective, and portable uranium sensory material with adequate selectivity is increasingly urgent and would be of great importance in environmental monitoring of radionuclides. Herein, we report a novel luminescent europium metal-organic framework (Eu-MOF) with plenty of Lewis basic sites for binding uranyl ions (UO22+), the most common form of uranium in solution, through a facile one-step solvothermal synthetic route. The mesoporous structure consists of europium nodes and flexible nitrogen-containing ligands with a 29.2 × 20.5 Å2 channel along the c-axis. Furthermore, the obtained material displays characteristic fluorescence of trivalent Eu3+ and could be applied as a turn-off sensory probe targeting UO22+ in solution. Differential fluorescent quenching occurred upon a series of potential interfering ions compared to UO22+ and the detection limit as low as 0.9 µM was achieved with a rapid response. Graphical abstract.

Lipidomic analysis of plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry.

Stroke is a major cause of mortality and long-term disability worldwide. The study of biomarkers and pathogenesis is vital for early diagnosis and treatment of stroke. In the present study, a continuous-flow normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF/MS) method was employed to measure lipid species in human plasma, including healthy controls and lacunar infarction (LI) patients. As a result, 13 lipid species were demonstrated with significant difference between the two groups, and a "plasma biomarker model" including glucosylceramide (38:2), phosphatidylethanolamine (35:2), free fatty acid (16:1), and triacylglycerol (56:5) was finally established. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 1.000 in the discovery set and 0.947 in the validation set for diagnosing LI patients from healthy controls. Besides, the sensitivity and specificity of disease diagnosis in validation set were 93.3% and 96.6% at the best cutoff value, respectively. This study demonstrates the promising potential of NP/RP 2D LC-QToF/MS-based lipidomics approach in finding bio-markers for disease diagnosis and providing special insights into the metabolism of stroke induced by small vessel disease. Graphical abstract Flow-chart of the plasma biomarker model establishment through biomarker screening and validation.

Study on the interaction of uranyl with sulfated beta-cyclodextrin by affinity capillary electrophoresis and molecular dynamics simulation.

The study on sulfated beta-cyclodextrin binding to uranyl ion helps to get a better understanding of uranyl compounds' intermolecular interaction mechanism and facilitates the structure-based design of uranyl binding molecules. Here we investigated the electromigration of the inclusion complex by using affinity capillary electrophoresis in acidic solution. The binding constant was determined to be logK = 2.96 ± 0.02 (R2 = 0.996) through nonlinear regression approach. The possible configurations and structural features of the inclusion complex were further studied by molecular dynamics simulation. The results suggest the distinctions of coordination environment and hydration compared with bare uranyl ion in aqueous solution. Thus, two water oxygen atoms coordinated with uranyl in the first hydration shell at 2.55 angstrom instead of five in the same distance range. The binding free energy was calculated as -12.10 ± 1.46 kcal/mol by means of thermodynamic perturbation method. The negative value indicates that the process of S-ß-CD capture uranyl ion in the aqueous media is spontaneous.

Binding constant determination of uranyl-citrate complex by ACE using a multi-injection method.

The binding constant determination of uranyl with small-molecule ligands such as citric acid could provide fundamental knowledge for a better understanding of the study of uranyl complexation, which is of considerable importance for multiple purposes. In this work, the binding constant of uranyl-citrate complex was determined by ACE. Besides the common single-injection method, a multi-injection method to measure the electrophoretic mobility was also applied. The BGEs used contained HClO4 and NaClO4 , with a pH of 1.98 ± 0.02 and ionic strength of 0.050 mol/L, then citric acid was added to reach different concentrations. The electrophoretic mobilities of the uranyl-citrate complex measured by both of the two methods were consistent, and then the binding constant was calculated by nonlinear fitting assuming that the reaction had a 1:1 stoichiometry and the complex was [(UO2 )(Cit)](-) . The binding constant obtained by the multi-injection method was log K = 9.68 ± 0.07, and that obtained by the single-injection method was log K = 9.73 ± 0.02. The results provided additional knowledge of the uranyl-citrate system, and they demonstrated that compared with other methods, ACE using the multi-injection method could be an efficient, fast, and simple way to determine electrophoretic mobilities and to calculate binding constants.