An ultra-robust fingerprinting method for quality assessment of traditional Chinese medicine using multiple reaction monitoring mass spectrometry

Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale fingerprinting method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.

Multiple reaction monitoring for the detection of disease-related synaptic proteins

Synaptic dysfunction occurs early in Alzheimer＇s disease （AD） and is acknowledged as a primary pathologic target for treatment. Synaptic degeneration is the pathological feature most strongly correlated with loss of cognitive function ante mortern （Terry et al., 1991）. Synapses are heavily damaged in hippocampal and neocortical regions of AD brain, whereas motor and occipital cortices are relatively spared （Honer et al., 1992）. Despite extensive work, the molecular mechanisms underlying synaptic degeneration are largely unknown.

Rapid Simultaneous Determination of Four Ganoderic Acids in Ganoderma(Chinese Name:Lingzhi)by Direct Infusion-Multiple Reaction Monitoring Cubed

Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the measurement workload for a huge number of testing samples.Hence,it is urgent to pursue more efficient approaches for the quality evaluation.Because of the greater selectivity of MRM cubed(MRM^(3))over MRM,there might be a chance to omit the time-intensive LC separation.In current study,we attempted to develop a direct infusion(DI)-MRM^(3) program,and the applicability was thereafter assessed through simultaneous determination of four ganoderic acids(GAs)in one of the most famous tonic herbal medicines namely Ganoderma(Chinese name:Lingzhi).Primary parameters such as Q1>Q3>QLIT ion transitions,collision energy(CE),and excitation energy were optimized by programming online energy-resolved mass spectrometry with authentic compounds.A single DI-MRM measurement merely costed four minutes,and in spite of the wide occurrences of isomers,satisfactory selectivity was achieved.Method validation assays demonstrated the method to be sensitive,precise,accurate,and reproducible.The quantitative results from DI-MRM^(3) were also justified by conducting LC-MRM measurements in parallel.Significant differences occurred for the content patterns between the two original sources namely Ganoderma lucidum and G.sinense,and,moreover,either cultivar or harvest time showed dramatical influence on the quantitative features of the four targeted GAs.More importantly,DI-MRM3 is a meaningful analytical option for rapid quantitative analysis of herbal medicines,because of the comparable reliability,nonetheless,less consumptions of both measurement time and solvent,compared with LC-MRM.

Quantitative determination of multi-class bioactive constituents for quality control of Yiqi Jiangzhi Granules

Objective:To establish a reliable and sensitive method for evaluating quality of Yiqi Jiangzhi Granules(YQJZG).Methods:Ultra performance liquid chromatography electrospray ionization tandem mass spectrometry(UPLC-ESI-MS/MS)was employed for simultaneous determination of eight marker components.Separation was performed on an AQUITY UPLC;HSS T3 column,the mobile phase consisted of acetonitrile as the organic phase and 0.1%(volume percentage)formic acid as the aqueous.Eight marker components,ginsenoside Rg1(GRg1),ginsenoside Re(GRe),ginsenoside Rb1(Gb1),typhaneoside(TEO),isorhamnetin-3-O-neohespeidoside(IN),hesperidin(HPD),aurantio-obtusin-6-O-β-D-glucoside(AG)and curcumin(CCM),were detected by multiple reaction monitoring(MRM)mode.The Chinese Pharmacopoeia(2020 edition)was regarded as the guidance document for this method validation.Results:The method showed good linearity(R^(2)≥0.9990).The relative standard deviation(RSD)values for the instrument precision,intermediate precision and repeatability were less than 2.91%,2.88%,and 3.54%,respectively.The average recovery varied from 91.08%to 103.89%,with RSD below 3.81%.Sample solutions were found to be stable within 24 h at 4℃(RSD<2.85%).Eight marker components were successfully determined from three batches of YQJZG.Conclusion:The proposed UPLC-ESI-MS/MS method was found to be simple,fast and sensitive,and can be used for the routine quality assessment of YQJZG.Simultaneously,this method may provide a new and powerful tool of quality control for other traditional Chinese medicine analogous formulae.

Quantitative Evaluation of Aldo-keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo-keto reductases （AKRs） in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measure- ments of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring （MRM） of mass spectrometry （MS） to examine the protein expression of AKRs in five different hepatic cell lines. These include one rela- tively normal hepatic cell line, L-02, and four hepatocellular carcinoma （HCC） cell lines, HepG2, HUH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HUH7. Similar observations were obtained through MRM. Different from HUH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distri- bution of AKR 1C proteins is likely to be associated with liver tumorigenesis, and the AKR expres- sion status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HUH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein fam- ily at both mRNA and protein levels were feasible using current techniques.

Atmospheric pressure gas chromatography-tandem mass spectrometry analysis of fourteen emerging polycyclic aromatic sulfur heterocycles in PM_(2.5)

Research on pollution characteristics and toxicities of emerging polycyclic aromatic sulfur heterocycles(PASHs) in PM_(2.5) has not been reported due to the lack of analytical method with the needed performance.In the present study,a novel method for the determination of 14 PASHs in PM_(2.5) was developed using atmospheric pressure gas chro matography-tandem mass spectrometry(APGC-MS/MS).Atmospheric pressure chemical ionization was operated with multiple reaction monitoring in positive ionization mode.High sensitivity(method detection limit <1.673 pg/m^(3)),acceptable re coveries(67.6%-120.8%) and precisions(RSD of 2.2%-15.4%) were obtained.The method was successfully applied for analyzing PASHs in 10 PM_(2.5) samples collected from Taiyuan,a typical industrial city in China,in 2016,The total concentrations were from 929 pg/m^(3) to 14,593 pg/m^(3).The determined levels indicated that further investigations on environmental fate and toxicities of PM_(2.5)-bound PASHs may be needed.

Multiresidue pesticide analysis in tomato using GC-MS/MS using modified QuEChERS method with titanium-coated graphite with CNT-ABS nanocomposite as dispersive solid-phase extraction materials

The presence of pesticide residues in food and vegetables is a growing concern for consumers.To monitor these residues reliably,a selective and sensitive multiresidue system has been developed and validated in tomato by gas chromatography–tandem mass spectrometry(GC-MS/MS).Titanium-coated graphite with carbon nanotube(CNT)in acrylonitrile–butadiene–styrene(ABS)used as reversed-dispersive solid-phase extraction materials with modified QuEChERS(Quick,Easy,Cheap,Effective,Rugged,and Safe)method.Titanium-coated graphite with CNT-ABS is synthesized,characterized by X-ray diffraction and scanning electron microscopy.The clean-up performance of titanium-coated graphite with CNT-ABS was demonstrated to be better to primary secondary amine and graphitized carbon black cartridges.The processing of two multiple reaction monitoring transformations for each analyte is done using GC-MS/MS in electron impact mode.Satisfactory purification and recovery effects(74%–100%)of 35 pesticides were achieved in tomato matrices when using 5 mg of titanium-coated graphite ABS-CNT nanocomposite.It was observed that quinolphos and deltamethrin are present above the maximum residue limit.The technique proved to be reliable and sensitive in tomato samples for the routine testing of 35 pesticides.