Proteogenomic analysis identifies neoantigens and bacterial peptides as immunotherapy targets in colorectal cancer.

Considerable progress has recently been made in cancer immunotherapy, including immune checkpoint blockade, cancer vaccine, and adoptive T cell methods. The lack of effective targets is a major cause of the low immunotherapy response rate in colorectal cancer (CRC). Here, we used a proteogenomic strategy comprising immunopeptidomics, whole exome sequencing, and 16 S ribosomal DNA sequencing analyses of 8 patients with CRC to identify neoantigens and bacterial peptides that can serve as antitumor targets. This study directly identified several personalized neoantigens and bacterial immunopeptides. Immunoassays showed that all neoantigens and 5 of 8 bacterial immunopeptides could be recognized by autologous T cells. Additionally, T cell receptor (TCR) αß sequencing revealed the TCR repertoire of epitope-reactive CD8+ T cells. Functional studies showed that T cell receptor-T (TCR-T) could be activated by epitope pulsed lymphoblastoid cells. Overall, this study comprehensively profiled the CRC immunopeptidome, revealing several neoantigens and bacterial peptides with potential to serve as immunotherapy targets in CRC.

CovPDB: a high-resolution coverage of the covalent protein-ligand interactome.

In recent years, the drug discovery paradigm has shifted toward compounds that covalently modify disease-associated target proteins, because they tend to possess high potency, selectivity, and duration of action. The rational design of novel targeted covalent inhibitors (TCIs) typically starts from resolved macromolecular structures of target proteins in their apo or holo forms. However, the existing TCI databases contain only a paucity of covalent protein-ligand (cP-L) complexes. Herein, we report CovPDB, the first database solely dedicated to high-resolution cocrystal structures of biologically relevant cP-L complexes, curated from the Protein Data Bank. For these curated complexes, the chemical structures and warheads of pre-reactive electrophilic ligands as well as the covalent bonding mechanisms to their target proteins were expertly manually annotated. Totally, CovPDB contains 733 proteins and 1,501 ligands, relating to 2,294 cP-L complexes, 93 reactive warheads, 14 targetable residues, and 21 covalent mechanisms. Users are provided with an intuitive and interactive web interface that allows multiple search and browsing options to explore the covalent interactome at a molecular level in order to develop novel TCIs. CovPDB is freely accessible at http://www.pharmbioinf.uni-freiburg.de/covpdb/ and its contents are available for download as flat files of various formats.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Advances in understanding the role of dendritic cells in aplastic anaemia.

Aplastic anaemia (AA) is an autoimmune disease characterized by haematopoietic failure in the bone marrow. Abnormal activation and hyperfunction of T lymphocytes are believed to cause bone marrow damage, which plays a major role in AA pathogenesis. Dendritic cells (DCs) play a vital role in the immune system by processing antigens for presentation to T cells and regulating their differentiation and function. DC dysfunction may cause abnormal T-cell activation. Recent studies have associated the occurrence and development of AA with DC function. In this review, we have discussed the role of DCs in AA pathogenesis and their potential as putative therapeutic targets for AA.

Promoted Generation of T Helper 1-Like Regulatory T Cells After Transient Middle Cerebral Artery Occlusion in Type-2 Diabetic Mice.

BACKGROUND: Regulatory T cells (Tregs) play a remarkable role in modulating post-ischemic neuroinflammation. However, the characteristics of Tregs in diabetic ischemic stroke remain unknown. METHODS: Transient middle cerebral artery occlusion (MCAO) was conducted on leptin receptor-mutated db/db mice and db/+ mice. The number, cytokine production, and signaling features of Tregs in peripheral blood and ipsilateral hemispheres were evaluated by flow cytometry. Treg plasticity was assessed by the adoptive transfer of splenic Tregs into mice. The effect of ipsilateral macrophages/microglia on Treg plasticity was determined by in vitro co-culture analysis. RESULTS: db/db mice had more infiltrating Tregs in their ipsilateral hemispheres than db/+ mice. Infiltrating Tregs in db/db mice expressed higher transforming growth factor-ß (TGF-ß), interleukin-10 (IL-10), forkhead box P3 (Foxp3), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and T-box expressed in T cells (T-bet) in comparison to infiltrating Tregs in db/+ mice, suggesting promoted generation of T helper 1 (Th1)-like Tregs in the brains of db/db mice after stroke. The post-ischemic brain microenvironment of db/db mice significantly up-regulated IFN-γ, TNF-α, T-bet, IL-10, and TGF-ß in infiltrating Tregs. Moreover, ipsilateral macrophages/microglia remarkably enhanced the expression of IFN-γ, TNF-α, and T-bet but not IL-10 and TGF-ß in Tregs. db/db macrophages/microglia were more potent in up-regulating IFN-γ, TNF-α, and T-bet than db/+ macrophages/microglia. Interleukin-12 (IL-12) blockage partially abolished the modulatory effect of macrophages/microglia on Tregs. CONCLUSION: The generation of Th1-like Tregs was promoted in the brains of type 2 diabetic mice after stroke. Our study reveals significant Treg plasticity in diabetic stroke.Abbreviations: Foxp3: forkhead box P3; IFN-γ: interferon-γ; IL-10: interleukin-10; IL-12: interleukin-12; MCAO: middle cerebral artery occlusion; PBS: phosphate-buffered saline; STAT1: Signal transducer and activator of transcription 1; STAT5: Signal transducer and activator of transcription 1; T-bet: T-box expressed in T cells; TGF-ß: transforming growth factor-ß; Th1: T helper 1; TNF-α: tumor necrosis factor-α; Tregs: regulatory T cells. Foxp3: forkhead box P3; IFN-γ: interferon-γ; IL-10: interleukin-10; IL-12: interleukin-12; MCAO: middle cerebral artery occlusion; PBS: phosphate-buffered saline; STAT1: Signal transducer and activator of transcription 1; STAT5: Signal transducer and activator of transcription 1; T-bet: T-box expressed in T cells; TGF-ß: transforming growth factor-ß; Th1: T helper 1; TNF-α: tumor necrosis factor-α; Tregs: regulatory T cells.

StreptomeDB 3.0: an updated compendium of streptomycetes natural products.

Antimicrobial resistance is an emerging global health threat necessitating the rapid development of novel antimicrobials. Remarkably, the vast majority of currently available antibiotics are natural products (NPs) isolated from streptomycetes, soil-dwelling bacteria of the genus Streptomyces. However, there is still a huge reservoir of streptomycetes NPs which remains pharmaceutically untapped and a compendium thereof could serve as a source of inspiration for the rational design of novel antibiotics. Initially released in 2012, StreptomeDB (http://www.pharmbioinf.uni-freiburg.de/streptomedb) is the first and only public online database that enables the interactive phylogenetic exploration of streptomycetes and their isolated or mutasynthesized NPs. In this third release, there are substantial improvements over its forerunners, especially in terms of data content. For instance, about 2500 unique NPs were newly annotated through manual curation of about 1300 PubMed-indexed articles, published in the last five years since the second release. To increase interoperability, StreptomeDB entries were hyperlinked to several spectral, (bio)chemical and chemical vendor databases, and also to a genome-based NP prediction server. Moreover, predicted pharmacokinetic and toxicity profiles were added. Lastly, some recent real-world use cases of StreptomeDB are highlighted, to illustrate its applicability in life sciences.

Digital measurement and correlation analysis of coronal and sagittal anatomic parameters in the radiographs of adolescent patients with idiopathic scoliosis.

OBJECTIVE: To use digital software to measure the morphologic and anatomic parameters of adolescent idiopathic scoliosis (AIS). Differences and correlations among different parameters were compared to provide an anatomic basis for the selection of treatment methods and preoperative evaluation of AIS. METHODS: Spinal radiographs were taken from 300 boys and girls (age, 10-18 years) suffering from idiopathic scoliosis in four grade-A hospitals in Inner Mongolia. After screening, 120 cases with complete imaging data were assessed. Imaging data were transferred to a work station (Dr Wise™). The anatomic indices of the Cobb Angle, CVA, AVT, TS, CA, CPT, CSI, FPT, CCA, TK, LL, SS, PT, and PI were measured. RESULTS: There were significant differences in AVT between different grades and types of scoliosis (F = 34.079, P = 0.000; χ2 = 23.379, P = 0.000). AVT was a protective factor, and the smaller the AVT, the less severe was the scoliosis. Compared with adolescents with mild or moderate scoliosis, the Cobb angle of adolescents with severe scoliosis was negatively correlated with CCA, LL, and SS (r = - 0.641, p < 0.05; r = - 0.695, p < 0.01; r = - 0.814, p < 0.01). CONCLUSIONS: Some of the anatomic parameters in the coronal and sagittal planes of adolescents with idiopathic scoliosis were significantly different according to the severity and type of scoliosis. Significant correlations were found between more anatomic indices in adolescents with severe scoliosis than in adolescents suffering from mild or moderate scoliosis.

HyperCys: A Structure- and Sequence-Based Predictor of Hyper-Reactive Druggable Cysteines.

The cysteine side chain has a free thiol group, making it the amino acid residue most often covalently modified by small molecules possessing weakly electrophilic warheads, thereby prolonging on-target residence time and reducing the risk of idiosyncratic drug toxicity. However, not all cysteines are equally reactive or accessible. Hence, to identify targetable cysteines, we propose a novel ensemble stacked machine learning (ML) model to predict hyper-reactive druggable cysteines, named HyperCys. First, the pocket, conservation, structural and energy profiles, and physicochemical properties of (non)covalently bound cysteines were collected from both protein sequences and 3D structures of protein-ligand complexes. Then, we established the HyperCys ensemble stacked model by integrating six different ML models, including K-nearest neighbors, support vector machine, light gradient boost machine, multi-layer perceptron classifier, random forest, and the meta-classifier model logistic regression. Finally, based on the hyper-reactive cysteines' classification accuracy and other metrics, the results for different feature group combinations were compared. The results show that the accuracy, F1 score, recall score, and ROC AUC values of HyperCys are 0.784, 0.754, 0.742, and 0.824, respectively, after performing 10-fold CV with the best window size. Compared to traditional ML models with only sequenced-based features or only 3D structural features, HyperCys is more accurate at predicting hyper-reactive druggable cysteines. It is anticipated that HyperCys will be an effective tool for discovering new potential reactive cysteines in a wide range of nucleophilic proteins and will provide an important contribution to the design of targeted covalent inhibitors with high potency and selectivity.

Liquid-solid ratio during hydrothermal carbonization affects hydrochar application potential in soil: Based on characteristics comparison and economic benefit analysis.

Returning straw-like agricultural waste to the field by converting it into hydrochar through hydrothermal carbonization (HTC) is an important way to realize resource utilization of waste, soil improvement, and carbon sequestration. However, the large-scale HTC is highly limited by the large water consumption and waste liquid pollution. Here, we propose strategies to optimize the liquid-solid ratio (LSR) of HTC, and comprehensively evaluate the stability, soil application potential, and economic benefits of corn stover-based hydrochar under different LSRs. The results showed that the total amount of dissolved organic carbon of hydrochars increased by 55.0% as LSR reducing from 10:1 to 2:1, while the element content, thermal stability, carbon fixation potential, specific surface area, pore volume, and functional group type were not obviously affected. The specific surface area and pore volume of hydrochar decreased by 61.8% and 70.9% as LSR reduced to 1:1, due to incomplete carbonization. According to the gray relation, hydrochar derived at LSR of 10:1 and followed by 2:1 showed greatest relation degree of 0.80 and 0.70, respectively, indicating better soil application potential. However, reducing LSR from 10:1 to 2:1 made the income of single process production increased from -388 to 968 ¥, and the wastewater generation decreased by 80%. Considering the large-scale application of HTC in fields for farmland improvement and environmental remediation, the comprehensive advantages of optimized LSR will be further highlighted.

A multi-stage ensemble network system to diagnose adolescent idiopathic scoliosis.

OBJECTIVES: To develop a deep learning algorithm to automatically evaluate and diagnose scoliosis on full spinal X-ray images. METHODS: This retrospective study collected full spinal X-ray images (anteroposterior) from four hospital databases from January 1, 2018, to March 31, 2021. The data were divided into training and validation sets. Full spinal X-ray images for external validation were independently collected at one hospital from April 1, 2021, to June 30, 2021. Model effectiveness was validated with a public dataset. Statistical software R was used to analyze the accuracy and sensitivity of the model curvature and anatomical balance parameters and assess interrater consistency. RESULTS: This study included 788 and 185 training and test datasets, respectively. The accuracy and recall of the algorithm model for the Cobb angle, apical vertebrae (AV), upper vertebrae, and lower vertebrae were 89.36%, 85.71%, 77.2%, and 80.24% and 97.35%, 93.38%, 84.11%, and 87.42%, respectively. The symmetric mean absolute percentage error at the Cobb angle was 5.99%, and the automatic measurement time was 1.7 s. The mean absolute error values of the Cobb angle and the distances between the center sacral vertical line and AV and C7 plumb line were 1.07° and 1.12 and 1.38 mm, respectively. Statistical analysis confirmed that the Cobb angle results were in good agreement with the gold standard (interclass coefficients of 0.996, 0.978, and 0.825; p < 0.001). CONCLUSION: Our deep learning algorithm model had high sensitivity and accuracy for scoliosis, which could help radiologists improve their diagnostic efficiency. KEY POINTS: • Our deep learning algorithm model had high sensitivity and accuracy for scoliosis, which could help radiologists improve their diagnostic efficiency. • Multi-center validation data were used in this study to guarantee the reliability of the research. • Algorithmic model measures 200 times faster than radiologists.

Sex differences in arterial identity correlate with neointimal hyperplasia after balloon injury.

BACKGROUND: Endovascular treatment of atherosclerotic arterial disease exhibits sex differences in clinical outcomes including restenosis. However, sex-specific differences in arterial identity during arterial remodeling have not been described. We hypothesized that sex differences in expression of the arterial determinant erythropoietin-producing hepatocellular receptor interacting protein (Ephrin)-B2 occur during neointimal proliferation and arterial remodeling. METHODS AND RESULTS: Carotid balloon injury was performed in female and male Sprague-Dawley rats without or 14 days after gonadectomy; the left common carotid artery was injured and the right carotid artery in the same animal was used as an uninjured control. Arterial hemodynamics were evaluated in vivo using ultrasonography pre-procedure and post-procedure at 7 and 14 days and wall composition examined using histology, immunofluorescence and Western blot at 14 days after balloon injury. There were no significant baseline sex differences. 14 days after balloon injury, there was decreased neointimal thickness in female rats with decreased smooth muscle cell proliferation and decreased type I and III collagen deposition, as well as decreased TNFα- or iNOS-positive CD68+ cells and increased CD206- or TGM2-positive CD68+ cells. Female rats also showed less immunoreactivity of VEGF-A, NRP1, phosphorylated EphrinB2, and increased Notch1, as well as decreased phosphorylated Akt1, p38 and ERK1/2. These differences were not present in rats pretreated with gonadectomy. CONCLUSIONS: Decreased neointimal thickness in female rats after carotid balloon injury is associated with altered arterial identity that is dependent on intact sex hormones. Alteration of arterial identity may be a mechanism of sex differences in neointimal proliferation after arterial injury.

Breeding a Soybean Cultivar Heinong 531 with Peking-Type Cyst Nematode Resistance, Enhanced Yield, and High Seed-Oil Contents.

Soybean cyst nematode (SCN) is a destructive threat to soybean production. It is economically important to develop a new SCN-resistant soybean cultivar with high yield and other good agronomic traits. In this study, a yellow-seed-coated and yellow-hilum-pigmented cultivar Heinong 531 belonging to maturity group I was developed by a pedigree breeding method through a test-cross between a female parental SCN-resistant soybean cultivar Pengdou 158 and a male parental line F1 (high-yield but SCN-susceptible Hefeng 55 × SCN-resistant Kangxian 12). Heinong 531 was evaluated for SCN resistance in both SCN-infested field and autoclaved soil inoculated with hatched second-stage juveniles of SCN HG Type 0. The results indicated that SCN development at all stages in Heinong 531 was suppressed and the female index was only 1.6 to 5.6%. Heinong 531 as well as Pengdou 158 and Kangxian 12 were identified as carrying the Peking-type resistance with both rhg1-a GmSNAP18 and Rhg4 GmSHMT08 genes. In the 2-year regional trials, the average yield of Heinong 531 reached 2805.0 kg/ha, and the 1-year production trial demonstrated an average yield of 2,751.5 kg/ha with yield increase of >12.0% when compared with the local cultivars. The average seed-fat (oil) contents of Heinong 531 reached up to 22.3%. The Peking-type SCN-resistant Heilong 531 with enhanced yield and high seed-oil contents was released in China in June 2021 with the certified number of 'Heishendou 20210004'. These agronomic traits make Heinong 531 a good prospect in a wide attempt to control SCN in the main soybean-producing areas of Northeast China.

Pre-procedural color duplex ultrasound evaluation predicts restenosis after long-segment superficial femoral artery stenting.

OBJECTIVES: Restenosis after stenting for superficial femoral artery atherosclerotic disease remains a significant clinical problem, especially for long-segment lesions. We assessed predictors of in-stent restenosis in patients with long-segment superficial femoral artery disease and hypothesized that pre-procedural ultrasound assessment would predict in-stent restenosis. METHODS: This single-center study retrospectively analyzed 283 limbs in 243 patients who treated with superficial femoral artery nitinol stent placement for long-segment (≥15 cm) lesions between 2015 and 2018. Color duplex ultrasound was performed pre-procedure and post-procedure at 3, 6, 12, 24, and 36 months. The endpoint was ≥50% in-stent restenosis in the superficial femoral artery. Primary patency rates were analyzed with Kaplan-Meier survival analysis and compared using the log-rank test. A multivariable Cox proportional hazards model was used to evaluate the risk factors for in-stent restenosis. RESULTS: The median length of lesions was 25.8 ± 8.1 cm. The cumulative freedom from ≥50% in-stent restenosis at 3, 6, 12, 24, and 36 months was 95.3%, 78.3%, 56.0%, 30.6%, and 15.9%, respectively. Univariate and multivariate Cox regression analysis showed that cumulative lesion length ≥ 25 cm (hazard ratio 1.681; p = 0.003), calcified plaque (hazard ratio 1.549, p = 0.006), poor runoff scores >10 (hazard ratio 1.870, p = 0.003), and chronic renal failure (hazard ratio 2.075, p = 0.009) were independent risk factors for in-stent restenosis. The agreement rate between ultrasound and angiography was 92.6% for cumulative lesion length (κ 0.851) and 91.9% for runoff score (κ 0.872). CONCLUSIONS: The results indicate that pre-procedural color duplex ultrasound evaluation is helpful for the selection of appropriate candidates for superficial femoral artery stent placement. Cumulative lesion length ≥25 cm, plaque calcification, poor distal runoff, and chronic renal failure independently predicted in-stent restenosis.

Design, Synthesis, Biological Evaluation and Silico Prediction of Novel Sinomenine Derivatives.

Sinomenine is a morphinan alkaloid with a variety of biological activities. Its derivatives have shown significant cytotoxic activity against different cancer cell lines in many studies. In this study, two series of sinomenine derivatives were designed and synthesized by modifying the active positions C1 and C4 on the A ring of sinomenine. Twenty-three compounds were synthesized and characterized by spectroscopy (IR, 1H-NMR, 13C-NMR, and HRMS). They were further evaluated for their cytotoxic activity against five cancer cell lines, MCF-7, Hela, HepG2, SW480 and A549, and a normal cell line, Hek293, using MTT and CCK8 methods. The chlorine-containing compounds exhibited significant cytotoxic activity compared to the nucleus structure of sinomenine. Furthermore, we searched for cancer-related core targets and verified their interaction with derivatives through molecular docking. The chlorine-containing compounds 5g, 5i, 5j, 6a, 6d, 6e, and 6g exhibited the best against four core targets AKT1, EGFR, HARS and KARS. The molecular docking results were consistent with the cytotoxic results. Overall, results indicate that chlorine-containing derivatives might be a promising lead for the development of new anticancer agents.

Graphics processing unit accelerating compressed sensing photoacoustic computed tomography with total variation.

Photoacoustic computed tomography with compressed sensing (CS-PACT) is a commonly used imaging strategy for sparse-sampling PACT. However, it is very time-consuming because of the iterative process involved in the image reconstruction. In this paper, we present a graphics processing unit (GPU)-based parallel computation framework for total-variation-based CS-PACT and adapted into a custom-made PACT system. Specifically, five compute-intensive operators are extracted from the iteration algorithm and are redesigned for parallel performance on a GPU. We achieved an image reconstruction speed 24-31 times faster than the CPU performance. We performed in vivo experiments on human hands to verify the feasibility of our developed method.

Simultaneous determination and pharmacokinetics study of four quinones in rat plasma by ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry after the oral administration of Qianzhi capsules.

A sensitive, specific, and accurate ultra high-performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantification of purpurin, munjistin, mollugin, and alizarin from Qianzhi capsules in rat plasma. Chromatographic separation was performed on an Agilent Eclipse Plus C18 RRHD column with a mobile phase consisting of methanol and 5 mM ammonium acetate/water with gradient elution. The analytes were quantified on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode and switching the electrospray ion source polarity with positive electrospray ionization in a single run. Samples were pretreated by liquid-liquid extraction with cyclohexane. The intra- and interday precision and accuracy of the assay were within acceptable ranges. Matrix effects for all of the analytes were between 90.16 and 100.21%. The average recovery ranged from 75.38 to 88.96%. This method was successfully applied to study the pharmacokinetic parameters of the four compounds in rat plasma after oral administration of Qianzhi capsules. Four quinones could be rapidly absorbed into blood (tmax , 0.80-1.93 h) and eliminated relatively slowly (t1/2 , 8.07-11.97 h). The results might be helpful for guiding the clinical application of Qianzhi capsules in the future.

On the Sparse Beamformer Design.

In designing acoustic broadband beamformers, the complexity can grow significantly when the number of microphones and the filter length increase. It is advantageous if many of the filter coefficients are zeroes so that the implementation can be executed with less computation. Moreover, the size of the array can also be pruned to reduce complexity. These problems are addressed in this paper. A suitable optimization model is proposed. Both array pruning and filter thinning can be solved together as a two-stage optimization problem to yield the final sparse designs. Numerical results show that the complexity of the designed beamformers can be reduced significantly with minimal effect on performance.

Simultaneous Determination of Seven Phenolic Acids in Rat Plasma Using UHPLC-ESI-MS/MS after Oral Administration of Echinacea purpurea Extract.

A rapid and sensitive Ultra High Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (UHPLC-ESI-MS/MS) method was developed and validated to simultaneously determine the concentration of seven phenolic acids (syringic acid, ferulic acid, caffeic acid, vanillic acid, p-coumaric acid, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid) in rat plasma after oral administration of Echinacea purpurea extract. After mixing with the internal standard (IS), butylparaben, plasma samples were prepared by liquid-liquid extraction with ethyl acetate. The separation was performed using the Agilent Eclipse Plus C18 column (1.8 µm, 2.1 mm × 50 mm) with a gradient system consisting of solution A (0.1% acetic acid in water) and solution B (methanol) at a flow rate of 0.3 mL/min. The detection was accomplished by a multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). The method was validated in terms of linearity, precision, accuracy, extraction recovery, matrix effect and stability. This method was successfully applied to study the pharmacokinetic properties of the seven compounds after oral administration of Echinacea purpurea extract in rats.

Determination and Pharmacokinetic Study of Three Diterpenes in Rat Plasma by UHPLC-ESI-MS/MS after Oral Administration of Rosmarinus officinalis L. Extract.

Rosmarinus officinalis L. is commonly used as a spice and flavoring agent. Diterpenes are the main active compounds of R. officinalis. An Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-ESI-MS/MS) method was developed for the determination of carnosol, rosmanol, and carnosic acid isolated from R. officinalis in rat plasma, and applied to a pharmacokinetic study after oral administration of R. officinalis extract. Sample preparation involved a liquid-liquid extraction of the analytes with ethyl acetate. Butylparaben was employed as an internal standard (I.S.). Chromatographic separation was carried out on a C18 column (ACQUITY UPLC® HSS T3, 1.8 µm, 2.1 mm × 100 mm) with a gradient system consisting of the mobile phase solution A (0.1% formic acid in water) and solution B (acetonitrile) at the flow rate of 0.3 mL/min. The quantification was obtained using multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). The UHPLC-MS/MS assay was validated for linearity, accuracy, precision, extraction recovery, matrix effect and stability. This study described a simple, sensitive and validated UHPLC-MS/MS method for the simultaneous determination of three diterpene compounds in rat plasma after oral administration of R. officinalis extract, and investigated on their pharmacokinetic studies as well.