CH11 Analysis of Indeterminate Structures by the Flexibility Method

SOD1基因p.H44R罕见位点突变致肌萎缩侧索硬化症1例报告并文献复习王雅欢，杨偲，刘洪雨，何金婷，王姣琦摘要：目的探讨SOD1基因常见突变位点的临床特点，为肌萎缩侧索硬化症（ALS）的早期识别、诊断及病程评估提供帮助。

方法回顾性分析1例SOD1基因第二外显子c.131A>G:p.H44R突变致ALS患者的临床资料及基因检测结果，并结合文献讨论。

结果本例患者以右下肢疼痛、无力伴肌肉萎缩起病，感觉系统无阳性体征，肌电图提示未受累肢体出现亚临床的神经源性改变，经全外显子测序发现SOD1基因第二外显子c.131A>G:p.H44R突变，该变异为罕见变异。

结论ALS早期诊断困难，不同基因位点突变致ALS的临床表现存在差异。

基因检测可辅助诊断，在疾病早期具有一定鉴别意义。

关键词：肌萎缩侧索硬化；运动神经元病；临床分型；基因型中图分类号：R744.8 文献标识码：AAmyotrophic lateral sclerosis caused by a rare mutation in the SOD1 gene at p. H44R locus： a case report and lit⁃erature review WANG Yahuan，YANG Si，LIU Hongyu，et al.（Fourth Inpatient Area of Department of Neurology，China-Japan Union Hospital of Jilin University， Changchun 130033， China）Abstract：Objective This study aims to explore the clinical characteristics of common mutation sites in the SOD1 gene and provide assistance for the early identification, diagnosis, and course evaluation of amyotrophic lateral sclerosis (ALS). Methods The clinical data and genetic testing results of a patient with ALS caused by the c.131A>G:p.H44R muta⁃tion in the second exon of the SOD1 gene were retrospectively analyzed and discussed in conjunction with the literature. Results The patient presented with pain and weakness in the right lower limb accompanied by muscle atrophy. No posi⁃tive signs were observed in the sensory system. The electromyogram revealed subclinical neurogenic changes in the unaf⁃fected limbs. Whole-exome sequencing identified a rare mutation in exon c.131A>G:p.H44R of the SOD1gene. Conclusion Early diagnosis of ALS is challenging, and the clinical manifestations vary depending on the gene site muta⁃tions. Genetic testing can assist in diagnosis and has significant identification value in the early stages of the disease.Key words：Amyotrophic lateral sclerosis；Motor neuron disease；Clinical classification；Genotype运动神经元病（motor neuron disease，MND）是一组起病隐匿的慢性进行性神经系统变性疾病，病因尚不十分清楚，可能与遗传或环境因素相关。

Modern Research in Catalysis, 2012, 1, 11-14doi:10.4236/mrc.2012.12002 Published Online July 2012 (/journal/mrc)TsOH·H2O-Catalyzed Friedel-Crafts of Indoles of3-Hydroxyisobenzofuran-1(3H)-One with Indoles: Highly Synthesis of 3-Indolyl-Substituted PhthalidesHongying Tang, Xinyu Zhang, Airu Song, Zhongbiao Zhang*Tianjin Key Laboratory of Water Resources and Environment, Tianjin Normal University, Tianjin, ChinaReceived April 5, 2012; revised May 3, 2012; accepted May 22,2012ABSTRACTAn efficient and facile method for the synthesis of 3-indolyl-substituted phthalides by Friedel-Crafts alkylation of in-doles with 3-hydroxyisobenzofuran-1(3H)-one has been developed. Using only 2 mol-% TsOH·H2O as the catalyst, various substituted indoles can react smoothly at room temperature to give the corresponding phthalides products in good to excellent yields (up to 96%).Keywords: Synthesis; 3-Indolyl-Substituted Phthalides; Friedel-Crafts Alkylation; 3-Hydroxyisobenzofuran-1(3H)-One;Indole1. IntroductionCurrently, the chemistry of phthalides has attracted much attention from many research groups, as they are the key skeleton for a number of synthetic and naturally occurr- ing bioactive molecules [1-5]. In particular, 3-substituted phthalide moieties are embodied in numerous natural pr- oducts. For examples, (-)-Alcyopterosin E which contai- ns phthalides bone shows mild cytotoxicity toward Hep-2 (human larynx carcinoma) cell line [6]; 3-Butylphthali- des isolated from the basidiomycete Phanerochaete velut- ina CL6387 appear to be specific for Helicobacter pylori [7]; and Cytosporone E has antifungal activities [8]; Fus- cinarin isolated from soil fungas was found to compete effecttively with macrophage inflammatory protein (MIP)-1αfor binding to human CCR5, an important anti HIV-1 target that interferes with HIV entry into cells [9]. On the other hand, the indole framework is a privileged structure motif in a large number of natural products and therapeutic agents [10,11]. The history of researches in indole and its derivatives has been more than 100 years, and this domain has become one of the hot research spot. And with the fresh and rapid development of life scien- ces, this research of indoles has drawn increasing atten- tion from organic chemists [12-15]. Furthermore, it is well known that there are more than 3000 kinds of indole derivatives in the nature. Among them, more than 40 kinds are treatment medicines [11]. The potential biolog- ical activities of phthalides and indoles promoted us to develop a synthesis of an interesting type of heterocyclic compounds via their combination. And it probably offers great opportunities for the lead compound discovery. However, due to the phthalides’ specific structure feature containing the activity lactone motif, the methodology for the synthesis of 3-indolylsubstituted phthalides has been rarely explored. Up to date, the synthesis of this type compounds is mainly restricted to the reaction of indoles with 2-forylbenzoic acids either at high tempera-ture (T > 100˚C) [16-18] or in the presence of an acidic cation exchange resin Amberlyst 15 [19]. The main drawbacks associated with these previously reported pro- cedures are the unavailability of the common starting materials, 2-forylbenzoic acids, and in some cases, harsh reaction conditions [20-23]. Therefore, the development of simple, highly efficient alternative approach for the synthesis of 3-indolyl substituted phthalides is highly desirable. Herein, we report an efficient substitution re-action of 3-hydroxyisobenzofuran-1(3H)-one [24-26], which can be easily prepared via the reduction of readily available phthalic anhydride, and indoles using the TsOH·H2O as a catalyst at room temperature for the syn-thesis of 3-indolyl substituted phthalides with high yields.2. Experimental2.1. General Information1H and 13C NMR spectra were recorded in CDCl3or DMSO on a Bruker AMX-300 or Varian 400 MHz instr- umental using TMS as an internal standard. Elemental*Corresponding author.H. Y. TANG ET AL. 12analyses were conducted on a Yanaco CHN Corder MT- 3 automatic analyzer. Melting points were determineed on a T-4 melting point apparatus. All temperatures were uncorrected.All solvents and chemicals were commercially avail-able and used without further purification unless other-wise stated.3-Hydroxyisobenzofuran-1(3H)-OneAccording to the literature [26,27], the solution of isob- enzofuran-1(3H)-one [25] 1.34 g (1 mmol), NBS 2.71 g (1.2 mmol), AIBN 0.03 g (0.2 mmol) in 15 mL CCl4 was heated to reflux until isobenzofuran-1(3H)-one disap- peared (monitored by TLC). After removal of the sol- vent, the residue was purified by column chromatogram- phy on silica gel (200 - 300 mesh, gradient elution with petroleum ether/ethyl acetate = 61) to afford 3-brom- oisobenzofuran-1(3H)-one 1.81 g, (85%). 3-bromoiso- benzofuran-1(3H)-one was heated to reflux in water for 2 h, then the mixture was cooled to room temperature, a white crystal was separated out, after filtration, the prod- uct 3-hydroxyisobenzofuran-1(3H)-one was got, 1.13 g (88%), mp. 98˚C.2.2. General Procedure for the Synthesis of3-Indolyl-Substituted Phthalides Catalyzedby TsOH·H2OThe solution of TsOH·H2O (0.02 mmol), 3-hydroxyiso- benzofuran-1(3H)-one (1.00 mmol), and indoles (1.20 mmol) in CHCl3 (2 mL) was stirred at ambient tempera- ture until the raw material disappeared (monitored by TLC). After removal of the solvent, the residue was puri- fied by column chromatography on silica gel (200 - 300 mesh, gradient elution with petroleum ether/ethyl acetate = 31) to afford the product.3a-3h, 3j, 3l and 3n are known products according to the literatures [16,18,19].2.2.1. 3-(5-Hydroxy-1H-indol-3-Yl)isobenzofuran-1(3H)-One (3i)White solid, yield: 89%, mp. 213.4˚C - 215.1˚C. 1H NMR (DMSO-d6, 400 MHz) δ:5.07 (s, OH), 6.56 (d, J = 8.0 Hz, 1 H), 6.78 (s, 1 H), 6.88 - 6.90 (m, 1 H), 7.10 (d, J = 7.6 Hz, 1 H), 7.29 (d, J = 8.0 Hz, 1 H), 7.50 (d, J = 7.6 Hz, 1 H), 7.62 (t, J = 7.2 Hz, 1 H), 7.80 (t, J = 7.2 Hz, 1 H), 8.08 (d, J = 7.6 Hz, 1 H), 10.18 (s, NH); 13C NMR (DMSO-d6, 100 MHz) δ: 171.2, 154.6, 150.2, 135.9, 132.3, 131.5, 129.8, 129.3, 128.0, 127.5, 124.5, 120.2, 114.3, 112.9, 105.5, 78.5; C16H11NO3 (265.07): calcd. C 72.45, H 4.18, N 5.28; found C 72.70, H 3.99, N 5.16. 2.2.2. 3-(6-(Benzyloxy)-1H-indol-3-Yl)isobenzofuran-1(3H)-One (3k)White solid, yield: 89%, mp. 194.5˚C - 195.7˚C. 1H NMR (CDCl3, 400 MHz), δ:5.19 (d, J = 3.2 Hz, 2H, CH2), 6.60 - 6.68 (m, 2 H), 6.84 (s, 1 H), 6.92 (d, J = 2.8 Hz, 1 H), 7.26 - 7.98 (m, 9 H), 8.00 (d, J = 8.8 Hz, 1 H), 10.24 (b, NH). 13C NMR (CDCl3, 100 MHz), δ:170.6, 154.3, 149.4, 137.7, 135.0, 132.3, 129.5, 128.6, 127.9, 127.6, 126.6, 126.1, 125.8, 125.3, 123.3, 122.9, 119.0, 113.0, 108.6, 82.5, 70.4; C23H17NO3 (355.12): calcd. C 77.73, H 4.82, N 3.94; found C 77.57, H 4.99, N 3.98.2.2.3.3-(6-Chloro-1H-indol-3-Yl)isobenzofuran-1(3H)-One (3m)White solid, yield: 88%, mp. 139.1˚C - 140.6˚C. 1H NMR (DMSO-d6, 400 MHz), δ:6.77 (d, J = 3.6 Hz, 1H), 6.91 (s, 1H), 7.04 (dd, J = 2.8 Hz, 9.2 Hz, 1 H),7.52-7.68 (m, 5H), 7.96 (d, J = 9.6 Hz, 1H), 8.83 (s, 1 H); 13C NMR (DMSO-d6, 100 MHz) δ:170.9, 150.7, 137.7, 135.6, 130.3, 128.7, 128.2, 127.4, 125.9, 124.4, 123.6, 119.4, 114.5, 111.9, 78.6; C16H10ClNO2 (283.04): calcd.C 67.74, H 3.55, N 4.94; found C 67.84, H 3.56, N 4.86.3. Results and DiscussionIn our initial studies, all kinds of experimental parame- ters, such as solvents, molar ratios of the two substrates and catalyst loadings, were thoroughly investigated in the model Friedel-Crafts alkylation of indole (2a) with3-hydroxyisobenzofuran-1(3H)-one (1) employing the TsOH·H2O as the catalyst. And the results are listed in Table 1.Solvent evaluation revealed that chloroalkanes (CH2Cl2 and CHCl3) favored this transformation in termsof high yield (Table 1, Entries 1 and 2), and CH2Cl2 isTable 1. Optimization of the reaction conditions a.EntryCatalyst(mol-%)SolventMolar ration(1:2)Time(h)Yield(%)b1 2 CHCl3 1:1.2 3 902 2 CH2Cl2 1:1.23 953 2 THF 1:1.2 3814 2 EA 1:1.2 3725 2 CH3OH 1:1.2 5 616 2 EtOH 1:1.2 5617 1 CH2Cl2 1:1.2 12 788 5 CH2Cl2 1:1.2 2 949 2 CH2Cl2 1.2:1 3 8310 2 CH2Cl2 1:1 3 8711 2 CH2Cl2 1:1.5 3 92a Reactions were performed on a 1 mmol scale of 1(3-hydroxyisobenzo- furan-1(3H)-one) with a concentration of 0.5 mol·L–1;b Isolated yield.H. Y. TANG ET AL.13the optimal solvent providing the corresponding Friedel- Crafts alkylation product with the highest yield of 95% (Table 1, Entry 2, 95%). Marked decrease in yields was observed in this reaction when using other solvents, such as THF, methanol, ethanol and ethyl acetate (Table 1, Entries 3-6). Moreover, it was found that a ring-opened product was obtained in the case of the methanol or ethanol as the solvent. In addition, the adjustment of the catalyst loading also brought out some influence both on the reaction rate and chemical yield. For example, using the great amount of catalyst, we could obtain the alkyla-tion product 3a in quite shorter time with almost the same high level yield (Table 1, Entry 8). Reducing the catalyst loading from 2 mol-% to 1 mol-% led to an ob-vious decrease both on reaction rate and yield. Further-more, the molar ratio of the two reactants was found to be an essential factor to the yield of the reaction. As shown in Table 1, the yield of 3a was enhanced with the decrease in the molar ratio of 1 to 2a, and the maximal yield of 95% was obtained when the molar ratio reached 1:1.2 (Table 1, Entry 2). Further decreased the molar ratio to 1:1.5 resulted in a lower yield of 3a(Table 1, Entry 10).On the basis of the optimal reaction conditions of in- dole 2a (2 mol-% TsOH·H2O as the catalyst, substrates molar ratio (1:2a = 1:1.2), 0.5 M 3-hydroxyisobenzofu- ran-1(3H)-one 1, at 20in CH℃2Cl2), a plethora of in- doles 2 were evaluated for the reaction with 3-hydroxy- isobenzofuran-1(3H)-one 1, and the results are summa- rized in Table 2.As shown in Table 2, the reaction has broad applica- bility with respect to the indoles. A wide range of indoles bearing either an electron-withdrawing or electrondona- ting substituent at various positions on the indole ring were included as the reaction partners, leading to the formation of the desired products in good to excellent yields (Table 2, Entries 2-14, in most cases, over 90% yields were obtained). Generally, electron-rich indoles exhibited a higher reactivity than those of electron-poor ones. It is worth noting that the most electron-deficient nitro-substituted indole 2n also worked well to affording the corresponding alkylation product 3n with satisfactory yield (Table 2, Entry 14). Subsequently, the reaction be- tween indole (2a) and 3-ethyl-3-hydroxyisobenzofuran- 1(3H)-one [23,27], an interesting reaction partner, since an oxygen-containing quaternary carbon will be created in the Friedel-Crafts alkylation reaction, was also inves-tigated. Unfortunately, no reaction occurred even at ele-vated reaction temperature (60˚C).4. ConclusionIn summary, we have developed an efficient and facile method for the synthesis of 3-indolyl-substituted phthalides by Friedel-Crafts alkylation of indoles with Table 2.The synthesis of 3-indolyl-substituted phthalides between 3-hydroxyisobenzofuran-1(3H)-one and indoles catalyzed by TsOH·H2O a.Entry Product R R’ Time (h) Yield (%)b1 3a c H H 3 952 3b c H CH3 3 933 3c c 2-CH3 H 3 964 3d c 2-Ph H 3 885 3e c 5-OCH3H 3 966 3f c 5-OBn H 12 927 3g c 5-F H 24 918 3h c 5-Cl H 24 929 3i 5-OH H 24 8910 3j c 5-Br H 24 9411 3k 6-OBn H 12 8912 3l c 6-F H 5 8913 3m 6-Cl H 24 8814 3n c 6-NO2 H 24 77a Reactions were performed on a 1 mmol scale of 1 with a concentration of0.5 mol·L–1; b solated yield; c According to the literatures [16,18,19].3-hydroxyisobenzofuran-1(3H)-one. Compared to the limited literature reports, this method is more attractive because of its high efficiency, mild reaction conditions, readily available starting materials as well as the cheap catalyst. Various substituted indoles can react smoothly to give the corresponding phthalides in good to excellent yield. Attempts toward the asymmetric version of this alkylation reaction are underway in our laboratory at present.5. AcknowledgementsThis work is supported by the Research Fund for Young Scientist (No.52LX29), Tianjin.REFERENCES[1]T. K. Devon and A. I. Scott, “Handbook of NaturallyOccurring Compounds,” Vol. 1, Academic Press, NewYork, 1975.[2]J. B. John and S. C. Chou, “The Structural Diversity ofPhthalides from the Apiaceae,” Journal of Natural Prod-ucts, Vol. 70, No. 5, 2007, pp. 891-900.doi:10.1021/np0605586[3]R. Bentley, “Mycophenolic Acid: A One Hundred YearOdyssey from Antibiotic to Immunosuppressant,” Chemi-cal Reviews, Vol. 100, No. 10, 2000, pp. 3801-3826.[4]J. G. Lei, R. Hong, S. G. Yuan and G. Q. Lin, “Nickel-H. Y. TANG ET AL. 14Catalyzed Tandem. 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专利名称：预测癌症细胞对解旋酶抑制剂敏感性的方法专利类型：发明专利
发明人：坂本洋,加岛健史,小笠原清基,大手友贵,曾我真弓申请号：CN202080069044.3
申请日：20201120
公开号：CN114502746A
公开日：
20220513
专利内容由知识产权出版社提供
摘要：预测癌症细胞对解旋酶抑制剂敏感性的方法，其包括将检测到选自由TTK突变和RAD50突变组成的第1组的至少1种突变的癌症细胞预测为对解旋酶抑制剂具有敏感性的步骤；以及预测癌症细胞对解旋酶抑制剂敏感性的方法，其包括将检测到选自由RAD50突变、MRE11突变、NBN突变、DNA2突变和RBBP8突变组成的第2组的至少1种突变的癌症细胞预测为对解旋酶抑制剂具有敏感性的步骤。

申请人：中外制药株式会社
地址：日本国东京都
国籍：JP
更多信息请下载全文后查看。

Multiplicative effects model with internal standard in mobile phasefor quantitative liquid chromatography–mass spectrometryMi Song,Zeng-Ping Chen n,Yao Chen,Jing-Wen JinState Key Laboratory of Chemo/Biosensing and Chemometrics,College of Chemistry and Chemical Engineering,Hunan University,Changsha410082,Chinaa r t i c l e i n f oArticle history:Received10December2013Received in revised form8March2014Accepted12March2014Available online20March2014Keywords:Liquid chromatography–mass spectrometrySignal instabilityMultiplicative effects modela b s t r a c tLiquid chromatography–mass spectrometry assays suffer from signal instability caused by the gradualfouling of the ion source,vacuum instability,aging of the ion multiplier,etc.To address this issue,in thiscontribution,an internal standard was added into the mobile phase.The internal standard was thereforeionized and detected together with the analytes of interest by the mass spectrometer to ensure thatvariations in measurement conditions and/or instrument have similar effects on the signal contributionsof both the analytes of interest and the internal standard.Subsequently,based on the unique strategy ofadding internal standard in mobile phase,a multiplicative effects model was developed for quantitativeLC–MS assays and tested on a proof of concept model system:the determination of amino acids in waterby LC–MS.The experimental results demonstrated that the proposed method could efficiently mitigatethe detrimental effects of continuous signal variation,and achieved quantitative results with averagerelative predictive error values in the range of8.0–15.0%,which were much more accurate than thecorresponding results of conventional internal standard method based on the peak height ratio andpartial least squares method(their average relative predictive error values were as high as66.3%and64.8%,respectively).Therefore,it is expected that the proposed method can be developed and extendedin quantitative LC–MS analysis of more complex systems.&2014Elsevier B.V.All rights reserved.1.IntroductionMass spectrometry(MS)coupled with high performance liquidchromatography(HPLC)has become a widely used analyticaltechnique with its high sensitivity and high specificity[1–3].The role of the LC is to separate almost any mixture that can bedissolved,while the MS is to provide identification or quantitativedetermination by ionizing the separated peak.Currently,the mainapplication areas of LC–MS are in thefield of pharmaceutical,environmental and biochemical analysis[4].Déglon et al.[5]established an automated system applied to the pharmacokineticstudy offlurbiprofen(FLB)and its metabolite in human wholeblood without sample processing.Manfio et al.[6]developed amethod for simultaneous detection of sufentanil and morphine.Bassan et al.[7]quantitatively determined43common drugscontained in human serum.Due to its strong separation andstructural analysis capabilities,liquid chromatography tandem massspectrometry has also been widely applied to drug metabolism[8].Even though LC–MS has many excellent features,it also has itsown weak points.The interference of chemical background ions(chemical noise),signal drift,ion suppression,and the signalinstability limit its application in routine quantitative analysis.Many efforts have been made to overcome these problems.Forexamples,Guo et al.[9]used exclusive ion/molecule reactionswith dimethyl disulfide(DMDS)to reduce chemical noise.Autryet al.[10]coated a layer of gold on the surface of ion source toimprove signal stability.Annesley[11]confirmed that enhancedspecimen cleanup,chromatographic changes,reagent modifica-tions,and effective internal standardization could minimize orcorrect ion suppression.Nevertheless,the application of LC–MS inroutine quantitative analysis is still challenging.In quantitative analysis using LC–MS,the number of ionsdetected by mass spectrometry must be proportional to theamount of the analytes of interest injected.Hence,signal stabilityis of utmost importance for quantitative analysis using LC–MS.However,variations in either instrumental parts or measurementconditions can significantly influence the signal stability.It is wellknown that the key factor contributing to the signal instability inLC–MS is the ion source of mass spectrometer which is responsiblefor ionizing the injected analytes and further pushing the selectedions into the mass analyzer.The gradual fouling of the ion source,vacuum instability,and aging of the ion multiplier are likely tochange the ionization efficiency of the analytes of interest atdifferent times,and hence lead to signal instability.It wasContents lists available at ScienceDirectjournal homepage:/locate/talantaTalanta/10.1016/j.talanta.2014.03.0280039-9140/&2014Elsevier B.V.All rightsreserved.n Corresponding author.Tel./fax:þ8673188821989.E-mail addresses:zpchen2002@,zpchen@(Z.-P.Chen).Talanta125(2014)347–351observed in election impact ionization(EI)–quadrupole MS that a continuous loss of signal happened in10repeated injections with a more than50%decrease in peak areas after10injections.A maximum95%of the lost signal might be recovered after a time interval of at least32h.It should be emphasized that the above observation is a regular occurrence,rather than an occa-sional phenomenon[10].Continuous signal variations could inva-lidate the calibration models established for quantitative LC–MS analysis,if no proper measures have been taken to correct their detrimental effects.Consequently,calibration models need to be rebuilt frequently to ensure acceptable quantitative accuracy and precision.It is rather costly and time consuming.To overcome or minimize the detrimental effects of continuous signal variations on quantification results of LC–MS,an internal standard method[12,13]is generally adopted,where peak height ratios(or peak area ratios)of the peak of the analytes of interest to that of an internal standard are calculated and used in quantitative analysis.However,the presence of possible baseline drift and background interferences complicates the application of internal standard method.In addition,the difficulty infinding proper internal standards for complex systems also limits its application. Multiplicative calibration transfer[14,15]developed in area of NIR spectroscopy has been successfully utilized by Pavón et al.to rectify baseline drift and sensitivity changes over long periods of time in mass spectrometers[16].But one shortcoming of this method is that an extra set of samples must be analyzed at regular intervals.Therefore,the routine quantitative application of LC–MS still calls for more advanced methods which can eliminate the influence of baseline drift and sensitivity changes at minimum cost.In this paper,based on the multiplicative effects model devel-oped by Chen et al.[17–20]for quantitative spectroscopic analysis of complex systems involving solids,a unique method was proposed to address the problems caused by baseline drift and sensitivity changes,and hence realize the long term applicability of calibration models for quantitative LC–MS analysis.2.Novel quantitative strategy for LC/MS-multiplicative effects model with internal standard in mobile phase(MEM IS) For quantitative LC–MS analysis with continuous signal varia-tions(i.e.variations in sensitivity),the mass spectrum(x i,row vector)of the i th sample measured at the peak of the chromato-graphic elution curve of the target analyte can be expressed as follows:x i¼b i c targ;i s targþd i;i¼1;2;…;Nð1ÞHere,s targ and c targ;i are the pure mass spectrum and concentration of the target analyte in the i th sample,respectively;d i represents the possible baseline and background interferences in x i;N denotes the number of samples;b i accounts for the effects of variations in sensitivity on the mass spectrum of the i th sample, due to changes in measurement conditions(e.g.vacuum degree and environmental temperature)and/or ion suppression. Obviously,the relationship between c targ;i and x i does not follow a linear model because of the presence of the multiplicative parameter b i which varies across samples.To determine c targ;i accurately,the confounding effect of b i must be eliminated.The multiplicative effects model developed by Chen et al.[18] for quantitative spectroscopic analysis of complex systems reveals that multiplicative effects confounding with the concentrations of the target analyte can be estimated by optical path-length estima-tion and correction method—OPLEC[19,20]or its modification version[18,19]as long as another coexistent analyte with constant concentration underwent the same multiplicative confounding effects simultaneously.In quantitative LC–MS assays,one possible way to satisfy the above prerequisite is to add a small amount of certain internal standard chemical compound into the mobile phase.The internal standard added should not be retained on the solid phase of LC,and can be ionized and detected by mass spectroscopy.Consequently,the mass spectrum(x i)of the i th sample measured at the peak of the chromatographic elution curve of the target analyte contains the contributions of both the target analyte and internal standard in the mobile phase:x i¼b i Uðc targ;i U s targþc stand s standÞþd i;i¼1;2;…;Nð2ÞHere,c stand is the concentration of the internal standard added in the mobile phase,which is constant across samples;s stand denotes the pure mass spectrum of the internal standard.The multi-plicative parameter vector b(b¼[b1;b2;…;b N])for N calibration samples can be estimated from their mass spectra X cal(X cal¼[x1; x2;…;x N])by OPLEC or its modified version.Two calibration models can then be built by multivariate linear calibration meth-ods such as partial least squares(PLS)[21].Thefirst model is between X cal and b,and the other is between X cal and diag(c targ)b (diagðc targÞb¼½b1c targ;1;b2c targ;2;…;b N c targ;N ):b¼α11þX calβ1;diagðc targÞb¼α21þX calβ2ð3Þwhere1is a column vector and its elements equal unity.For simplicity,the same number of latent components is generally used in the above two PLS calibration models.Once the mass spectrum of a test sample at the peak of the chromatographic elution curve of the target analyte has been recorded,the multiplicative confound-ing effects caused by the variations in sensitivity can be removed by dividing the prediction of the second calibration model by the corresponding prediction of thefirst calibration model,and accu-rate concentration prediction for the target analyte in the test samples is therefore readily achieved according to Eq.(4):c targ;test¼α2þx testβ2α1þx testβ1ð4Þ3.Experimental3.1.Reagents and chemicalsNicotinamide(98.5%)was obtained from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China).Tyrosine(Tyr,99%)and Tryptophan(Trp,98%)were from Sigma-Aldrich(Shanghai,China). Phenylalanine(Phe,98%)was purchased from Shanpu Chemical Co.,Ltd.(Shanghai,China).HPLC grade methanol was from Oceanpak Alexative Chemical,Ltd.(Beijing,China).All of these products were used as received without further purification.Stock solution(0.1600μg/L)of each amino acid was prepared by dissol-ving an appropriate amount of corresponding amino acid in ultrapure water in25ml volumetricflasks at room temperature and stored at41C.A milli-Q system from Aquapro(Taiwan,China) was applied to produce ultra-pure water used throughout the experiment.3.2.Sample preparation for the determination of amino acids in waterAppropriate amounts of Tyr,Trp and Phe stock solutions were mixed and diluted with ultrapure water to prepare seven calibra-tion samples andfive test samples(hereinafter to be referred as “test set1”).Among the calibration samples,the concentrations of Tyr and Trp ranged from0.0100μg/ml to1.0000μg/ml,while in the test samples,the concentrations of Tyr and Trp were in the range of0.0400–0.8600μg/ml.The concentrations of Phe in bothM.Song et al./Talanta125(2014)347–351 348the calibration and test samples were kept constant (0.5000μg/ml).One week after the above twelve mixture samples were analyzed by LC –MS,another five test samples (hereinafter to be referred as “test set 2”)with different concentrations of Tyr and Trp were prepared and analyzed.The concentrations of Phe in the samples of test set 2were also kept at 0.5000μg/ml.3.3.HPLC –MS /MS analysisAll the samples were analyzed by a 1200HPLC system (Agilent Technologies)equipped with a C 18reversed-phase column (2.1mm i.d.Â150mm length)packed with 3.5μm particles (Agi-lent,USA).The column was maintained at 251C.The mobile phase used consisted of water containing 0.1%formic acid and 0.0500μg/ml nicotinamide (eluent A),and methanol (eluent B).The volume ratio of eluent A to eluent B was 50%:50%.The flow rate was 0.2ml/min.For each sample,a volume of 10μl was loaded onto the column via an auto-sampler from a 96-well sample tray.Mass spectra of samples were collected by an Agilent G6400Triple Quadrupole mass spectrometer (Agilent Technolo-gies)with electrospray interface (ESI)operated in the positive mode using the following settings:nebulizer pressure ¼15psi,capillary voltage ¼4000V,drying gas flow rate ¼11L/min;and drying gas temperature ¼3001C.The MS detector was operated in multiple reaction monitoring (MRM)mode at a rate of 1.23cycle/s.The chosen precursor ions and product ion transitions for Tyr,Trp and Phe were provided in Tables S1and S2(Supporting informa-tion).All the samples were analyzed three times.3.4.Data analysisThe estimation of the multiplicative parameter vector b in MEM IS for the calibration samples was estimated by OPLEC m [19].The performance of MEM IS was compared with the conventional internal standard method carried out by the quantitative analysis software provided by the HPLC –MS/MS instrument and the PLS calibration models in terms of root-mean-square error of prediction(RMSEP,RMSEP ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi∑N i ¼1ðc targ ;i À^c targ ;i Þ2=N q ),where ^c targ ;i is the predicted concentration for the target analyte in the i th sample)and the average relative prediction error (ARPE,ARPE ð%Þ¼100ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi∑N i ¼1j c targ ;i À^c targ ;i j =c targ ;i q ).4.Results and discussionFig.1a shows total ion chromatogram (TIC)of four samples measured on the same day.It can be seen that the peak heights of Tyr and Trp increase along with the increase of their concentra-tions.However,the peak height of Phe also shows signi ficant variation,despite its concentrations in the four samples being constant,which suggests that LC –MS signals are unstable even within very short time intervals.Increase in time interval resulted in obvious variations in both peak height and retention time of TIC (Fig.1b).More importantly,the degree of variations in peak heights of difference species differed signi ficantly.The peak height of Phe saw a prominent decrease,while that of Trp showed a slight increase.To overcome the lack of signal stability in quantitative LC –MS assays,the convention internal standard method was tried.Phe was chosen as the internal standard as its concentration was constant across samples.The raw mass spectra of the analytes of interest (i.e.,Trp and Tyr)recorded at the peaks of their chromato-graphic elution curves were divided by signal intensity at the mass to charge ratio of 120.1(which is most intense fragment ion of Phe).And then univariate calibration models were established between the signal intensity ratios at the most intense fragment ions of the analytes of interest and their concentrations in mixture samples.Table 1shows RMSEP and ARPE values for the calibration and test sets obtained by the univariate calibration models based on the conventional internal standard method.For the test samples analyzed on the same day as the calibration ones,the univariate calibration models gave rather accurate predictions,suggesting that the conventional internal standard method ef fi-ciently corrected the in fluence of signal instability within rela-tively short time intervals.However,as can be seen in Table 1,the univariate calibration models failed to provide satisfactory predic-tions for test set 2analyzed one week later.TheconcentrationFig.1.a)Total ion chromatograms (TICs)of four samples measured on the same day and b)TICs of the same sample measured on two different days with a time interval of aweek.The concentrations of Phe in all samples were 0.5000μg/ml.Table 1Recovery RMSEP and ARPE values for the calibration and test sets obtained by the univariate calibration models based on the conventional internal standard p.Calibration set Test set 1Test set 2RMSEP (μg/ml)ARPE (%)RMSEP (μg/ml)ARPE (%)RMSEP (μg/ml)ARPE (%)Trp 0.0012 2.10.0021 1.10.262166.3Tyr0.00574.20.00853.70.127828.3M.Song et al./Talanta 125(2014)347–351349predictions for Trp and Tyr in test set 2severely deviated from the actual values,with ARPE values of 66.3%and 28.3%,respectively.The failure of the conventional internal standard method can be explained by the differences in the degree of variations in peak heights of difference species.Therefore,the conventional internal standard method is unable to solve the problem of signal instabil-ity in quantitative LC –MS assays.To address the problem caused by the differences in the degree of variations in peak heights of difference species,nicotinamide with greater polarity than those of the analytes of interest was added into the mobile phase,ionized and detected together with the analytes of interest by the mass spectrometer.Variations in measurement conditions and/or instrument would then havesimilar effects on the signal contributions of both the analytes of interest and nicotinamide.Therefore,the mass spectra of samples measured at the peak of the chromatographic elution curve of the target analytes follow the MEM IS model in Eq.(2).The two multiplicative parameter vectors b for Tyr and Trp in the calibra-tion samples were estimated by OPLEC m and are displayed in Fig.2.Clearly,different samples had signi ficantly different multi-plicative parameters re flecting the signal instability across sam-ples.More interestingly,for most of the samples,the multiplicative parameters calculated for Tyr were different from the correspond-ing ones calculated for Trp,which further con firmed that varia-tions in measurement conditions and/or instrument might have different degrees of in fluence on the signals of different analytes with different retention times.Table 2shows the results of PLS and MEM IS calibration models.The RMSEP values of PLS calibration models for test set 2are far higher than the corresponding values for the test set rge deviations can readily be observed in the concentration predic-tions for both Trp and Try in some samples (Figs.3a and 4a).These results suggest that the PLS method is also not an effective way to deal with the problem of signal instability in quantitative LC –MS assays.Though the RMSEP values of MEM IS for test set 2are still somewhat larger than the corresponding values for test set 1,the predictive precision of MEM IS for test set 2is much more accurate in comparison with PLS.The concentrations of Tyr and Trp in the two test sets predicted by MEM IS are rather close to their expected values (Figs.3b and 4b).Such results are quite satisfactory,and demonstrate the effectiveness of MEM IS in correcting the in fluence of signal instability on quantitative results.5.ConclusionsLC –MS suffers from signal instability.Due to the differences in the degree of variations in signal intensities of difference species,the conventional internal standard method based on the peak height ratio (or peak area ratio)of the peak of the analytes of interest to that of an internal standard is unable to solve the problem of signal instability in quantitative LC –MS assays.With a view to effectively address the problem of signal instability,an internal standard which is not retained on the solid phase was added into the mobile phase,ionized and detected together with the analytes of interest by the mass spectrometer to ensure that variations in measurement conditions and/or instrument have similar effects on the signal contributions of both the analytesofFig.2.The multiplicative parameter vectors b for Tyr and Trp in the calibration samples estimated by OPLEC m .Table 2RMSEP and ARPE values for the test sets obtained by MEM IS and PLS p.MEM ISPLS Test set 1Test set 2Test set 1Test set 2RMSEP (μg/ml)APRE (%)RMSEP (μg/ml)APRE (%)RMSEP (μg/ml)APRE (%)RMSEP (μg/ml)APRE (%)Trp 0.02589.60.023614.60.031823.70.066164.8Tyr0.01558.10.042510.60.022510.20.111629.9Fig.3.Concentration predictions for Trp in both the test set 1(red circle)and test set 2(blue triangle)obtained by PLS (a)and MEM IS (b)calibration models.(For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)M.Song et al./Talanta 125(2014)347–351350interest and the internal standard.Based on the above unique strategy,a multiplicative effects model with internal standard in mobile phase (MEM IS )was developed for quantitative LC –MS assays.The experimental results on the determination of amino acids in water by LC –MS demonstrated that the continuous signal variation in LC –MS analysis could signi ficantly deteriorate the precision and accuracy of both the conventional internal standard method and partial least squares (PLS).The average relative predictive error values of conventional internal standard method and PLS were as high as 66.3%and 64.8%,respectively.In comparison,MEM IS could ef ficiently mitigate the detrimental effects of continuous signal variations,and achieved quantitative results with average relative predictive error values in the range of 8.0–15.0%.Therefore,it is reasonable to expect that MEM IS would become a promising alternative for quantitative LC –MS analysis.AcknowledgmentsThe authors acknowledge the financial support of the National Natural Science Foundation of China (Grant nos.21075034,and 21275046),the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no.20130161110027),and the Program for New Century Excellent Talents in University (NCET-12-0161).Appendix A.Supporting informationSupplementary data associated with this article can be found in the online version at /10.1016/j.talanta.2014.03.028.References[1]W.M.A.Niessen,Liquid Chromatography –Mass Spectrometry,2nd ed.,MarcelDekker,New York,1990(Chromatography Science Series).[2]W.M.A.Niessen,J.Chromatogr.A 1000(2003)413–436.[3]R.C.Willoughby,E.Sheehan,S.Mitrovich,A Global View of LC/MS:How toSolve Your Most Challenging Analytical Problems,Global View Publishing,Pittsburgh,1998.[4]W.M.A.Niessen,J.Chromatogr.A 856(1999)179–197.[5]J.Déglon, A.Thomas,Y.Daali, uer, C.Samer,J.Desmeules,P.Dayer,P.Mangin,C.Staub,J.Pharm.Biomed.Anal.54(2011)359–367.[6]J.L.Man fio,V.J.Santos,nchote,L.M.Santos,M.J.Carmona,J.O.Auler,L.B.Junior,C.M.Donaduzzi,S.R.C.J.Santos,J.AOAC Int.94(2011)136–142.[7]D.M.Bassan,F.Erdmann,R.Krüll,Anal.Bioanal.Chem.400(2011)43–50.[8]C.Fenselau,Annu.Rev.Pharmacol.Toxicol.32(1992)555–578.[9]X.H.Guo,A.P.Bruins,T.R.Covey,Anal.Chem.79(2007)4013–4021.[10]W.D.Autry,K.Wolfs,S.Yarramraju,A.V.Schepdael,J.Hoogmartens,E.Adams,Anal.Chem.82(2010)6480–6486.[11]T.M.Annesley,Clin.Chem.49(2003)1041–1044.[12]M.H.Hiatt,J.Chromatogr.A 1218(2011)498–503.[13]C.J.Baker,N.Y.Kovalskayae,N.M.Mocka,R.A.Owensa,K.L.Deahld,B.D.Whitakerb, D.P.Robertsc,R.W.Hammonda, A.A.Aver'yanov,Physiol.Mol.Plant Pathol.78(2012)31–37.[14]P.Geladi,b.60(2002)211–224.[15]R.N.Feundale,N.A.Woody,H.W.Tan,A.J.Myles,S.D.Brown,J.Ferré,b.64(2002)181–192.[16]J.L.P.Pavón,M.N.Sánchez,C.G.Pinto,espada,B.M.Cordero,Anal.Chem.75(2010)6361–6367.[17]Z.P.Chen,L.J.Zhong, A.Nordon, D.Littlejohn,M.Holden,M.Fazenda,L.Harvey,B.McNeil,J.Faulkner,J.Morris,Anal.Chem.83(2011)2655–2659.[18]Z.P.Chen,L.M.Li,J.W.Jin,A.Nordon,D.Littlejohn,J.Yang,J.Zhang,R.-Q.Yu,Anal.Chem.84(2012)4088–4094.[19]Z.P.Chen,J.Morris,E.Martin,Anal.Chem.78(2006)7674–7681.[20]J.W.Jin,Z.P.Chen,L.M.Li,R.Steponavicius,S.N.Thennadil,J.Yang,R.-Q.Yu,Anal.Chem.84(2012)320–326.[21]H.Martens,M.Martens,Multivariate Analysis of Quality:An Introduction,John Wiley &Sons,Chichester,UK,2001.Fig.4.Concentration predictions for Tyr in both the test set 1(red circle)and test set 2(blue triangle)obtained by PLS (a)and MEM IS (b)calibration models.(For interpretation of the references to color in this figure legend,the reader is referred to the web version of this 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小儿肺炎支原体肺炎并发消化道系统损害的相关影响因素分析*钱元原①　季卫刚①　陈艳艳①　张娟① 【摘要】　目的：探讨小儿肺炎支原体肺炎并发消化道系统损害的相关影响因素。

方法：回顾性分析2020年1月—2023年3月南通大学附属南通妇幼保健院收治的132例小儿肺炎支原体肺炎患儿临床资料，根据是否并发消化道系统损害分为消化道系统损害组（n=35）、非消化道系统损害组（n=97）。

收集患儿一般资料，包括性别、年龄、体重、发热病程、病程、大环内酯类药物开始使用时间、糖皮质激素开始使用时间、红细胞沉降率（ESR）、C反应蛋白（CRP）水平、白细胞（WBC）水平、中性粒细胞百分比。

对一般资料进行单因素分析，再对有统计学差异因素的进行多因素logistic回归分析。

结果：132例小儿肺炎支原体感染患儿发生35例消化道系统损害，发生率26.52%（35/132）。

单因素分析显示，两组性别、体重、病程、糖皮质激素开始使用时间、ESR、WBC、中性粒细胞百分比对比，差异均无统计学意义（P>0.05）。

两组年龄、发热病程、大环内酯类药物开始使用时间、CRP水平比较，差异均有统计学意义（P<0.05）。

logistic回归分析显示，年龄≤3岁、发热病程≥7 d、大环内酯类药物使用时间<3 d、CRP≥10 mg/L是小儿肺炎支原体感染并发消化道系统损害的独立危险因素（OR>1且P<0.05）。

结论：小儿肺炎支原体肺炎患儿并发消化道系统损害与年龄≤3岁、发热病程≥7 d、大环内酯类药物使用时间<3 d、CRP≥10 mg/L有关。

【关键词】　小儿肺炎支原体肺炎　消化道系统损害　影响因素　炎症水平　大环内酯类 Analysis of Related Influencing Factors of Digestive Tract System Damage in MycoplasmaPneumoniae Pneumonia in Children/QIAN Yuanyuan, JI Weigang, CHEN Yanyan, ZHANG Juan. //Medical Innovation of China, 2024, 21(09): 143-146 [Abstract]　Objective: To investigate the related influencing factors of digestive tract system damagein mycoplasma pneumoniae pneumonia in children. Method: Clinical data of 132 children with mycoplasmapneumoniae pneumonia admitted to Affiliated Maternity and Child Health Care Hospital of Nantong University fromJanuary 2020 to March 2023 were retrospectively analyzed, and they were divided into digestive system damagegroup (n=35) and non digestive system damage group (n=97) according to whether complicated with digestivetract system damage. General data of the children were collected, including sex, age, weight, fever course, diseasecourse, time when macrocyclic lactones began to be used, time when glucocorticoid began to be used, erythrocytesedimentation rate (ESR), C reactive protein (CRP) level, white blood cell (WBC) level, and the percentage ofneutrophil. Univariate analysis was carried out for the general data, and multivariate logistic regression analysis wascarried out for the factors with statistical difference. Result: Among 132 children with mycoplasma pneumoniaeinfection, 35 cases of digestive tract damage, the incidence rate was 26.52% (35/132). Univariate analysis showedthat gender, body weight, disease course, glucocorticoid initiation time, ESR, WBC, percentage of neutrophilswere compared between the two groups, the differences were not statistically significant (P>0.05). There werestatistically significant differences in age, course of fever, start time of macrocyclic lactones and CRP level betweenthe two groups (P<0.05). logistic regression analysis showed that age ≤3 years old, duration of fever ≥7 d, time ofmacrocyclic lactones drug use ≥3 d, CRP ≥10 mg/L were independent risk factors for mycoplasma pneumoniaeinfection complicated with digestive tract damage in children (OR>1 and P<0.05). Conclusion: Digestive tract systemdamage in children with mycoplasma pneumoniae pneumonia is associated with age ≤3 years old, duration of fever≥7 d, duration of macrocyclic lactones drug use ≥5 d, CRP ≥10 mg/L.*基金项目：2020年度南通市市级科技计划项目（JCZ20018）①南通大学附属南通妇幼保健院儿科　江苏　南通　226000通信作者：张娟- 143 - 肺炎支原体感染是小儿肺炎的常见病因，儿童身体免疫力与抵抗力低下，病菌容易入侵肺部，导致炎症发生[1-2]。

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有机化学英文文献翻译对称八溴代酞菁的合成及其特征K.R. Venugopala Reddy a,*, J. Keshavayya ba Department of Studies in Industrial Chemistry, Kuvempu University,Jnanasahyadri, Shankaraghatta - 577 451Shimoga District,Karnataka, Indiab Department of Studies in Chemistry, Kuvempu University,Jnanasahyadri, Shankaraghatta - 577 451Shimoga District,Karnataka, IndiaReceived 5 November 2001; received in revised form 14 December 2001;accepted 18 January 2002* Corresponding author. Tel.: +91-08282-56225; fax: +91-08282-37255.E-mail address: university@sancharnet.in (K.R. Venugopala Reddy).摘要现在已经提出一条既方便又简单合成对称1,3,8,10,15,17,22,24-溴代金属—Cu，Co，Ni，Zn酞菁颜料的路线。

金属酞菁是由相应的八氨基取代酞菁合成的。

合成的化合物经过元素分析、电子光谱、红外光谱、磁性测试、粉末X射线衍射实验和热重研究来评估其热稳性、结晶度、结构完整性和纯度。

经过讨论和分析发现取代基对于电子光谱的影响及轨道对磁矩的贡献远远超过了电子场强的影响。

关键词：酞菁八取代合成热度电子的颜料和染料1.引言酞菁在近几年引起广泛的关注不仅是因为酞菁的结构同一些能够维持生命的重要分子如叶绿素、血红素相似，而且它具有显著的上色功能。