Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.

Chemical Components of Achyranthes bidentata Leaves by Ultra High Performance Liquid Chromatography-Mass Spectrometry

[Objectives] To study the chemical components and relative content of Achyranthes bidentata leaves and provide a scientific basis for further development and utilization of A. bidentata leaves.[Methods] The chemical components of A. bidentata leaves were rapidly analyzed using the ultra high performance liquid chromatography-time of flight-high resolution mass spectrometry (UHPLC-TOF-MS).[Results] Thirty eight chemical compounds were identified in samples of A. bidentata leaves collected from Wen County of Henan Province, in which seven chemical compounds had the relative content higher than 5%, linoleic acid reached 25.7% and inokosterone A reached 13.8%.[Conclusions] A. bidentata leaves contain many kinds of chemical compounds. This study is expected to provide a certain basis for further extraction of linoleic acid and inokosterone A.

HPLC-MS/MS Determination of Oleandrin and Adynerin in Blood with Solid Phase Supported Liquid-Liquid Extraction

[Objectives]To optimize the determination method of oleandrin and adynerin in blood. [Methods]High performance liquid chromatography-mass spectrometry( HPLC-MS/MS) was applied to determine oleandrin and adynerin in blood. The blood sample was dispersed and fixed on a solid phase supported liquid-liquid extraction column and eluted with ethyl acetate. The resulting eluent was used for chromatographic separation with Kinetex C_(18) column as the separation column and gradient elution was performed using 10 mmol/L ammonium formate solution containing 0. 1%( volume fraction) formic acid and acetonitrile as the mobile phase. In the tandem mass spectrometry analysis,the detection was carried out using the electrospray positive ion source multiple reaction monitoring mode. [Results] The mass concentration of oleandrin and adynerin showed linear relationship in the range of 2-100 μg/L. The limit of detection( 3 S/N) of the method was 0. 5 μg/L.A blank sample was used as the substrate for the spike recovery test. The recovery rate was in the range of 90. 0%-98. 0%,and the relative standard deviation( RSD) of the measured values( n = 6) was in the range of 2. 1%-7. 3%. [Conclusions]The method established in this experiment has the benefits of simple pretreatment,good recovery,high sensitivity and strong specificity,and is expected to provide an ideal method for the determination of such drugs in blood.

‘桂葡6号’葡萄酒花色苷组分HPLC-MS分析

利用高效液相色谱-质谱联用技术测定野生资源‘桂葡6号’乙醇发酵结束和瓶储3个月的葡萄酒中花色苷类物质的组成及含量,并与欧亚种‘赤霞珠’进行对比。结果发现,‘桂葡6号’乙醇发酵结束和瓶储3个月酒样中花色苷总量为548.94 g/L和427.89 g/L,分别是同期‘赤霞珠’的1.2倍和2.7倍左右。‘桂葡6号’乙醇发酵结束和瓶储3个月酒样中分别检测到25种和19种花色苷,双糖苷是其中最主要的花色苷类型,含量最高的是甲基花青素-3,5-O-双葡萄糖苷。与欧亚种‘赤霞珠’相比,瓶储3个月‘桂葡6号’酒样中3’,5’-羟基取代花色苷比例较高,而酰基化、甲基化和吡喃型花色苷比例较低。‘桂葡6号’花色苷组成及含量与欧亚种‘赤霞珠’差异明显,这由其品种特性决定。

Determination of risperidone in human plasma by HPLC-MS/MS and its application to a pharmacokinetic study in Chinese volunteers

This study presents a rapid, specific and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay for determination of risperidone (RIS) in human serum using paroxetine as an internal standard (IS). An Alltima-C18 column (2.1 mm×100 mm, 3 μm) and a mobile phase consisting of 0.1% formic acid-acetonitrile (40:60, v/v) were used for separation. The analysis was performed by selected reaction monitoring (SRM) method, and the peak area of the m/z 411.3→191.1 transition for RIS was measured versus that of the m/z 330.1→192.1 transition for IS to generate the standard curves. The assay linearity of RIS was confirmed over the range 0.25～50.00 ng/ml and the limit of quantitation was 0.05 ng/ml. The linear range corresponds well with the serum concentrations of the analytes obtained in clinical pharmacokinetic studies. Intraday and interday relative standard deviations were 1.85%～9.09% and 1.56%～4.38%, respectively. The recovery of RIS from serum was in the range of 70.20%～84.50%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference products) in 18 healthy male Chinese volunteers. The result suggests that two formulations are bioequivalent.

Quantitative‑Profling Method of Serum Steroid Hormones by Hydroxylamine‑Derivatization HPLC-MS

A sensitive and rapid high performance liquid chromatography-mass spectrometry(HPLC-MS)method was developed and validated for simultaneous quantifcation of ten steroid hormones,including estrogens,androgens,progesterones,and corticosteroids four classes of steroids.The following ten steroid hormones were analyzed:progesterone,21-deoxycortisol,estrone,4-androstenedione,testosterone,dihydro-testosterone,androstenone,dehydroepiandrosterone,corticosterone and cortisone.Stable deuterated isotopes were used as internal standards for quantifcation.Sample preparation with and without derivatization were performed after liquid-liquid extraction,and the corresponding results were compared according to sensitivity and selectivity.Hydroxylamine derivatization was found to improve the ionization efciency of the analytes for electrospray ionization MS analysis.The gradient of mobile phase and experimental parameters for HPLC separation were optimized.The lower limits of quantifcation were in the range of 0.05-5 ng mL^(−1) with wide linear range for the ten steroid hormones.The intra-day precision<11.1%and recovery of 84.5-120% with negligible matrix efect were achieved,where within the acceptance limits of the FDA guideline.Total HPLC-MS analysis time was 6 min.This method enables simultaneous quantifcation of steroids in human serum.It will be helpful for the serum steroid profling in order to understand various endocrinology diseases.

Analysis of Polymer Impurities in Cephalosporin Antibiotics

[Objectives]To establish a HPLC-MS method for the determination of polymer impurities in cefathiamidine and its preparations.[Methods]Kromasil 100-5 C_(18) column(4.6 mm ×250 mm,5μm)was used for analysis;mobile phase ammonium acetate solution(pH 6.30)-acetonitrile,gradient elution;volumetric flow rate 1.0 mL/min;column temperature 40℃;multi-reaction monitoring mode was used for analysis,and positive ion scanning was chosen as the electrospray ion source.[Results]The resolution between impurities and main peaks under this method was greater than 1.5,and 8 known impurities and 2 polymer impurities could be completely separated and distinguish-ed.It was inferred that the molecular ion peak[M+H]^(+):m/z727.1874,m/z 785.1937 was the possible polymer impurity of this product.[Conclusions]A method for the analysis of polymer impurities in cefathiamidine and its preparations was formed,which could achieve the purpose of simultaneous analysis of small molecule impurities and polymer impurities,and could better control the content of single impurities in the polymer,providing a reliable inspection basis for strict control of cefathiamidine quality.

Mass Spectrometric Structure Elucidation of the Trivalent and Pentavalent Nitrogen Contaminants of Pholcodine in the Cough Relief Medical Form Tuxidrin

In the paper “Supercritical Fluid Chromatography-Mass Spectrometry (SFC-MS) and MALDI-TOF-MS of Heterocyclic Compounds with Trivalent and Pentavalent Nitrogen in Cough Relief Medical Forms Tuxi and Cosylan” [1], the presence of morphine and other degradation products of pholcodine in cough relief medical forms of Tuxi are discussed. Tuxiis recalled from the Norwegian market by Weifa pharmaceutical company, and hence it no longer presents problems to users and health authorities there;however, the medical form Tuxidrin, which contains a significant amount of pholcodine as the active pharmacological ingredient, is still marketed. In the present paper, Tuxidrin is analyzed to determine the presence of degradation products of pholcodine. The degradation of pholcodine to morphine has been discussed previously as a factor in the development of addiction to narcotics in young persons. The structures of the contaminants in Tuxidrin, such as oxides of pholcodine, are elucidated in the present paper. The toxicity and pharmacology of oxides of alkaloids have generally not been well studied, and very little is known about the toxicity and pharmacology of the degradation (oxidation) products of pholcodine: the N-oxide and the N, N'-dioxide of pholcodine. According to Brondz and Brondz[1], the N-oxide and possibly also the N, N'-dioxide are less toxic than the original alkaloids and possess greater pharmacological activity, and hence they may be a source of useful new semisynthetic drugs. The question of possible addiction to pholcodine oxides has not been studied, and the potential of these substances to provoke allergies is unclear. The recall of Tuxi from the Norwegian marketis mainly based on the fact that pholcodine causes significantly increased levels of IgE antibodies in sensitized patients. Tuxidrin contains pholcodine and has the same negative effect as Tuxi, namely provoking allergies or even anaphylactic shock. From this point of view, Tuxidrin has no advantage over Tuxi. These two medical forms only differ in one respect: Tuxidrin requires a prescription (prescription duty medicine), but Tuxi doesnot (prescription free medicine). This aspect is also discussed in the present paper.

Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressionsin Pancreas and Liver Tissues

Objective:To investigate the effects of Clean-DM1(C-DM1),a polyherbal formulation of Radix Scrophulariae,Radix Astragali,Rhizoma Atractylodis,and Radix Salviae Miltiorrhizae,on high-fat diet(HFD)-induced diabetes mice.Methods:The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis.Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry(HPLC-MS)analysis were conducted for quality control.For in vivo study,mice were induced diabetes by HFD for 12 weeks.The mice in the normal group(Nor)were maintained with a regular diet and treated with saline by gavage.The HFD model mice were randomly divided into 3 groups,including a HFD diabetic model group,a C-DM1 extract-administered group(C-DM1,500 mg/kg),and metformin-administered groups(Met,500 mg/kg),8 mice in each group.Food intake,body weight(BW),and fasting blood glucose(FBG)levels were recorded weekly for 4 weeks.After 4 weeks of treatment,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood glucose,low-density lipoprotein cholesterol(LDL-C)were determined using an automated clinical chemistry analyzer,and homeostatic model for assessing insulin resistance(HOMA-IR)levels and oral glucose tolerance test(OGTT)were detected.The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining.Insulin receptor substrate(IRS)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)and adenosine 5'-monophosphate-activated protein kinase(AMPK)expressions in liver and pancreas tissues were detected by Western blot analysis.Results:HPLC-MS identified dihydroisotanshinone,dihydroisotanshinone I,cryptotanshinone,harpagoside,and atractyloside A in C-DM1 extract.The administration of C-DM1 extract significantly decreased body weight,calorie intake,and the levels of blood glucose and insulin in the diabetic mice(P<0.05 or P<0.01).The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C,ALT and AST(P<0.01).The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice.The C-DM1 extract significantly increased the phosphorylation of IRS,AKT,and AMPK and the expression of PI3K in pancreas and liver tissues(P<0.05 or P<0.01),which was consistent with the analysis results of network pharmacology.Conclusion:C-DM1 extract improved diabetes symptoms in longterm HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues,suggesting that C-DM1 formulation may help prevent the progression of T2DM.

基于超高效液相色谱-质谱分析的代谢组学在小檗碱治疗多囊卵巢综合征研究中的应用

多囊卵巢综合征(polycystic ovary syndrome,PCOS)是妇女最常见的内分泌和代谢紊乱性疾病,合理的药物治疗非常重要。本研究中,超重/肥胖的多囊卵巢综合征患者经过小檗碱治疗3个月,采集治疗前后的血样进行分析,利用超高效液相色谱-质谱联用技术(UHPLC-MS)对治疗前后血清中的内源性物质进行了相对含量测定。正交信号校正的偏最小二乘模式识别分析结果显示治疗前后有明显的区分。多变量分析结合非参数检验找出的与小檗碱治疗相关的差异代谢物均与脂类代谢有关。临床生化数据结合代谢组学数据显示,小檗碱治疗超重/肥胖型的多囊卵巢综合征患者可以增加机体胰岛素敏感性、改善脂类代谢。上述研究结果表明代谢组学是研究中药治疗疾病效果的有效工具之一。

液相色谱-串联质谱法测定水环境中的十氯酮

建立了分析测定水环境中十氯酮的液相色谱-串联质谱法。水样经液液萃取、净化后，采用 Eclipse plus C18柱（100 mm×2.1 mm，3.5μm）分离，乙腈和水为流动相进行梯度洗脱，在电喷雾负离子多反应监测模式下进行检测，同位素内标法定量。结果表明：采用液相色谱-质谱联用技术，证实了十氯酮在甲醇中以半缩醛的形式存在，而在丙酮/乙腈中以偕二醇的形式存在。由于十氯酮极性较强，在净化时难以洗脱，并且不耐酸，所以不能与其他有机氯农药一起分析。十氯酮在5～100μg / L 范围有良好的线性关系，相关系数 r2=0.999，检出限及定量限分别为0.70 ng / L 和2.8 ng / L；在5、40和100 ng / L 3个浓度添加水平的平均回收率为95.1％～98.9％，相对标准偏差为3.85％～4.72％。本方法具有良好的灵敏度、回收率和重现性，适用于水环境中十氯酮的测定。

高效液相色谱-串联质谱法测定水产品中甲氧苄啶的残留

建立水产品中甲氧苄啶药物残留量测定的高效液相色谱-串联质谱方法。样品选择乙腈提取，用正己烷脱脂，固相萃取柱净化，采用LC-MS/MS选择反应监测（SRM）正离子模式测定进行定性、定量分析。结果表明：甲氧苄啶药物的检出限（LOD）为0.5μg/kg，定量限（LOQ）为1.0μg/kg，检测结果的相对标准偏差为1.78%-5.08%，加标回收率达到77.1%-93.5%。该方法具有比较高的重现性和选择性，在水产品中甲氧苄啶的残留测定中具有很好的应用前景。

生物表面活性剂鼠李糖脂的纯化与表征

生物表面活性剂鼠李糖脂是微生物在一定条件下产生的次级代谢产物,其分子具有极性亲水基团和非极性亲油基团结构,通常表现出很高的表面活性和界面优先分配能力。可靠的分离提纯方法和成分鉴定手段是鼠李糖脂生产工艺成功的重要保证。实验通过好氧发酵培养铜绿假单胞菌CCTCC AB93066、酸沉降分离得到鼠李糖脂后,利用柱色谱提纯技术得到纯化的鼠李糖脂的单糖脂和二糖脂,最后采用高效液相色谱-质谱联用法进行成分鉴定。结果显示这两种鼠李糖脂均含有3种主要成分,其中单糖脂的主要成分为RhaC10C10、RhaC10C12-H2、RhaC10C12,二糖脂的主要成分为Rha2C10C10、Rha2C10C12-H2、Rha2C10C12。该研究结果表明,铜绿假单胞菌CCTCC AB93066是一种良好的鼠李糖脂产生菌;酸沉降-柱色谱技术可以用于鼠李糖脂的深度提纯,且有较好的效果;而高效液相色谱-质谱联用技术对鼠李糖脂成分鉴定具有灵敏度高和准确性好等优点,是一种较为可靠的检测方法。

Pharmacokinetics of oxiracetam and its degraded substance (HOPAA) after oral and intravenous administration in rats

The pharmacokinetics of oxiracetam and its degraded substance(4-hydroxy-2-oxo-1-pyrrolidine acetic acid,HOPAA)after oral and intravenous administration in rats were studied using an established UPLC-MS/MS method.Three groups of rats after an overnight fasted received 10 g/kg(n=6)oxiracetam suspensions orally,and 2 g/kg(n=6)normal or degraded oxiracetam injections intravenously via a caudal tail vein,respectively.Before the pharmacokinetic experiment,a simple safety evaluation testwas conducted on the degraded oxiracetam injections containing 16.16% HOPAA in mice.There was no mortality by a single intravenous dose of 2 g/kg of degraded oxiracetam injections within twoweeks,demonstrating that HOPAA was non-toxic in mice.Following intravenous administration of the normal injections,the plasma concentration-time curves of oxiracetam and HOPAA both showed a rapid elimination phase.The values of t_(1/2)were 3.1±1.5 h for oxiracetamand 0.8±0.2 h for HOPAA,andthemean residencetimes(MRT)were 1.2±0.1h and 0.8±0.1h,respectively.Oxiracetam and HOPAA after intravenous administration of the degraded oxiracetam injections presented elimination patterns similar to those observed in the normal injections.Oral pharmacokinetic results showed that the Tmax was less than 1.5 h for the two analytes,and both had a longer t_(1/2) and MRT than those of intravenous administration.Contents of HOPAA in three groupswere calculated based on AUC_(0-t) values of the two analytes.The quantitative change of HOPAA in vivo was also evaluated by comparing the plasma concentrations of HOPAA and oxiracetamat the same time for every group.Additionally,the values of absolute bioavailability of oxiracetam were about 8.0%and 7.4%calculated by the normal or degraded oxiracetam injections,whichwere far less than the value of 75%reported in literature,indicating the necessity of further study.

Identification and Detection of Dyeing Components in Counterfeit Cinnabar (Elutriation)

[Objectives]This study aimed to analyze the dyeing status and dyeing components of counterfeit cinnabar(elutriation),in order to provide technical basis for cinnabar(elutriation)supervision.[Methods]The scarlet 808 in cinnabar(elutriation)was identified qualitatively using thin-layer chromatography(TLC)and high-performance liquid chromatography(HPLC),and the positive samples were confirmed by high-performance liquid chromatography-mass spectrometry(HPLC-MS).The conditions were as follows:column,Agilent Eclipse Plus C 18;mobile phase,acetonitrile-0.1%formic acid(85∶15);detection wavelength,520 nm;ion source,electrospray ion source;scanning mode,positive ion scanning;scanning range,100-700 m/z;cone voltage,3.5 kv;drying gas temperature,350℃;drying gas flow rate,12 L/min.[Results]The thin-layer chromatography spots were clear and separated well.Among the 13 batches of samples,12 batches were positive for scarlet 808.[Conclusions]The counterfeit cinnabar(elutriation)currently circulating in the market has dyeing problem,with scarlet 808 as the dye.The TLC,HPLC and HPLC-MS methods established in this article can quickly screen and confirm the scarlet 808 in cinnabar(elutriation).

Association of 2-methoxyestradiol levels with the occurrence and development of endometrial cancer in humans

Objective The aim of the study was to determine the association of urinary levels of estradiol(E_(2))and 2-methoxyestradiol(2-MeOE_(2))with the occurrence and development of endometrial cancer.Methods In this case-control study,24-h urine specimens were collected from 28 postmenopausal patients with endometrial cancer and 28 postmenopausal healthy female controls.The concentration of 2-MeOE_(2) was determined using liquid chromatography-mass spectrometry with hollow fiber liquid-phase microextraction.The concentration of E_(2) was determined using an enzyme-linked immunosorbent assay.Results Estrogen levels were different between the patients with endometrial cancer and controls.The relative quantity of E_(2) in the case group was higher than that in the control group(P<0.05),whereas that of 2-MeOE_(2) was lower in the case group than that in the control group(P<0.05).The ratio of E_(2)-to-2-MeOE_(2) in the case group was significantly higher than that in the control group(P<0.05).Conclusion The results of this study indicate an imbalance of estrogen metabolites in endometrial carcinogenesis.Reduced 2-MeOE_(2) levels and elevated E_(2)-to-2-MeOE_(2) ratio may be used as potential biomarkers for the risk assessment of estrogen-induced endometrial cancer.

Photocatalytic degradation of methyl orange using ZnO/TiO_(2) composites

ZnO/TiO_(2)composites were synthesized by using the solvothermal method and ultrasonic precipitation followed by heat treatment in order to investigate their photocatalytic degradation of methyl orange(MO)in aqueous suspension under UV irradiation.The composition and surface structure of the catalyst were characterized by X-ray diffraction(XRD),field emission scanning electron microscope(FE-SEM),and transmission electron microscopy(TEM).The degradation efficiencies of MO at various pH values were obtained.The highest degradation efficiencies were obtained before 30 min and after 60 min at pH 11.0 and pH 2.0,respectively.A sample analysis was conducted using liquid chromatography coupled with electrospray ionization ion-trap mass spectrometry.Six intermediates were found during the photocatalytic degradation process of quinonoid MO.The degradation pathway of quinonoid MO was also proposed.

Eco-friendly and Cleaner Process Using Online Microwave-assisted Steam Extraction Coupled with Solid-phase Extraction for Trace Analysis of Sulfonamides in Animal Feed

An environmentally friendly method for extracting sulfonamides（SAs） residues from animal feed was described and applied. The method used online microwave-assisted steam extraction coupled with solid phase extraction（MASE-SPE）, which was followed by the analysis using high performance liquid chromatography-mass spectro- metry（HPLC-MS/MS）. The SAs residues were extracted successively with water steam under microwave irradiation, and thus directly introduced into an SPE colunm containing cation-exchange resin. The SAs were then eluted with methanol-ammonia（90：10, volume ratio） from the SPE column and followed by HPLC-MS/IVIS. The limits of detec- tion（LODs） for the analytes ranged from 0.24 ng/g to 0.49 ng/g. The limits of quantification（LOQs） ranged from 0.82 ng/g to 1.63 ng/g. Average recoveries of SAs were 76.3%--92.1%. The developed method was a reliable and envi-ronmentally friendly alternative to previous methods with respect to time, solvent and labor consumption for the analysis of SAs in animal foodstuffs.

Determination of Diphenylamine in Gunshot Residue by HPLC‑MS/MS

A high performance liquid chromatography tandem mass spectrometry/mass spectrometry(HPLC-MS/MS)protocol was developed for the determination of diphenylamine(DPA).Four productions of DPA were selected for qualitative assay and the peak area of the main product ion for quantitation.By means of separation using an Agilent Extend-C18 column(CA,USA)(150 mm×4.6 mm,5μm)with methanol‑water(90:10)as the mobile phase,DPA was detected by electrospray ionization(ESI)tandem mass spectrometry in positive mode.The linearity of the peak area versus concentration ranged 5‑500 ng/mL,r^(2)=0.9978.The limit of detection(S/N=3)of this method was 0.3 ng/mL.This method is applicable for the determination of DPA in gunshot residue.