Tubastatin_A_Hydrochloride_LCMS_13855_MedChemExpress
洋甘菊提取液MSDS英文版
1. IDENTIFICATION OF THE SUBSTANCE/TREPARATION AND THE COMPANY/UNDERTAKING3.HAZARDS IDENTIFICATION4. FIRST AID MEASURESMATERIAL SAFETY DATA SHEETProduct name:Supplier:Tel:EMERGENCY OVERVIEW: May cause skin irritation and/or dermatitisPrinciple routes of exposure: Inhalation: Ingestion: Skin contact: Eye contact:SkinMay cause irritation of respiratory tract May be harmful if swallowed May cause allergic skin reaction Avoid contact with eyesStatements of hazard MAY CAUSE ALLERGIC SKIN REACTION.Statements of Spill of Leak Label Eliminate all ignition sources. Absorb and/or contain spill with inert materials (e.g., sand, vermiculite). Then place in appropriate container. For large spills, use water spray to disperse vapors, flush spill area. Prevent runoff from entering waterways or sewers.General advice:POSITION/INFORMATION ON INGREDIENTSInhalation:Skin contact:Ingestion:Eye contact:Protection of first – aiders:Medical conditions aggravated by exposure: In the case of accident or if you fell unwell, seek medical advice immediately (show the label where possible).Move to fresh air, call a physician immediately.Rinse immediately with plenty of water and seek medical adviceDo not induce vomiting without medical advice.In the case of contact with eyes, rinse immediately with plenty of water and seek medical advice.No information availableNone knownSuitable extinguishing media:Specific hazards:Special protective equipment for firefighters:Flash point:Autoignition temperature:NFPA rating Use dry chemical, CO2, water spray or “alcohol” foam Burning produces irritant fumes.As in any fire, wear self-contained breathing apparatus pressure-demand, MSHA/NIOSH (approved or equivalent) and full protective gearNot determinedNot determinedNFPA Health: 1 NFPA Flammability: 1 NFPA Reactivity: 0Personal precautions: Environmental precautions: Methods for cleaning up: Use personal protective equipment.Prevent product from entering drains.Sweep up and shovel into suitable containers for disposalStorage:7. HANDLING AND STORAGE5.FIRE-FIGHTING MEASURES6. ACCIDENTAL RELEASE MEASURESRoom temperature Handling:Safe handling advice: Incompatible products:Use only in area provided with appropriate exhaust ventilation.Wear personal protective equipment.Oxidising and spontaneously flammable productsEngineering measures: Respiratory protection: Skin and body protection:Eye protection: Hand protection: Hygiene measures:Ensure adequate ventilation.Breathing apparatus only if aerosol or dust is formed. Usual safety precautions while handling the product will provide adequate protection against this potential effect. Safety glasses with side-shieldsPVC or other plastic material glovesHandle in accordance with good industrial hygiene and safety practice.Melting point/range: Boiling point/range: Density: Vapor pressure: Evaporation rate: Vapor density: Solubility (in water): Flash point:Autoignition temperature:No Data available at this time. No Data available at this time. No data available No data available No data available No data available No data available Not determined Not determinedStability: Stable under recommended storage conditions. Polymerization: None under normal processing.Hazardous decomposition products: Thermal decomposition can lead to release of irritating gases and vapours such as carbon oxides.Materials to avoid: Strong oxidising agents.10. STABILITY AND REACTIVITY9. PHYSICAL AND CHEMICAL PROPERTIES8. EXPOSURE CONTROLS/PERSONAL PROTECTION11. TOXICOLOGICAL INFORMATIONConditions to avoid: Exposure to air or moisture over prolonged periods.Product information Acute toxicityChronic toxicity:Local effects: Chronic exposure may cause nausea and vomiting, higher exposure causes unconsciousness.Symptoms of overexposure may be headache, dizziness, tiredness, nausea and vomiting.Specific effects:May include moderate to severe erythema (redness) and moderate edema (raised skin), nausea, vomiting,headache.Primary irritation: Carcingenic effects: Mutagenic effects: Reproductive toxicity:No data is available on the product itself. No data is available on the product itself. No data is available on the product itself. No data is available on the product itself.Mobility:Bioaccumulation: Ecotoxicity effects: Aquatic toxicity:No data available No data available No data availableMay cause long-term adverse effects in the aquatic environment.12. ECOLOGICAL INFORMATION13. DISPOSAL CONSIDERATIONSWaste from residues/unused products:Contaminated packaging:Waste disposal must be in accordance with appropriate Federal, State and local regulations. This product, if unaltered by use, may be disposed of treatment at a permitted facility or as advised by your local hazardous waste regulatory authority. Residue from fires extinguished with this material may be hazardous.Do not re-use empty containers.UN/Id No:Not regulated14. TRANSPORT INFFORMATIONDOTProper shipping name: Not regulatedTGD(Canada)WHMIS hazard class: Non - controlledIMDG/IMOIMDG – Hazard Classifications Not ApplicableIMO – labels:15. REGULATORY INFOTMATION International Inventories16. OTHER INFORMATIONPrepared by: Health & SafetyDisclaimer: The information and recommendations contained herein are based upon tests believed to be reliable.However, XABC does not guarantee the accuracy or completeness NOR SHALL ANY OF THIS INFORMATION CONSTITUTE A WARRANTY, WHETHER EXPRESSED OR IMPLIED, AS TO THE SAFETY OF THE GOOD, THE MERCHANTABILITY OF THE GOODS, OR THE FITNESS OF THE FITNESS OF THE GOODS FOR A PARTICULAR PURPOSE. Adjustment to conform to actual conditions of usage maybe required. XABC assumes no responsibility for results obtained or for incidental or consequential damages, including lost profits arising from the use of these data. No warranty against infringement of any patent, copyright or trademark is made or implied.End of safety data sheet。
高效液相色谱法检测（S）-2-氨基丁酰胺盐酸盐的纯度
高效液相色谱法检测（S）-2-氨基丁酰胺盐酸盐的纯度郝玉红;朱力敏;薛韧婕【摘要】用高效液相色谱法定量分析(S)-2-氨基丁酰胺盐酸盐的纯度。
用Agilent 1100型高效液相色谱仪，色谱柱Xterra C18（5µm，4.6 mm×250 mm），流动相为20%乙腈-80%，缓冲液：1000 mL水+5.0 g庚烷磺酸钠；检测波长为210 nm，流量为0.7 mL•min-1，柱温30℃，对(S)-2-氨基丁酰胺盐酸盐纯度进行定量测定。
相对标准偏差0.15%，标准回收率99.8%~100.2%，方法重复性好，定量准确度高。
%Purity analysis of (S)-2-Aminobutyramide hydrochloride was established by high performance liquid chromatography with Agilent 1100, Xterra C18(5 μm, 4.6 mm×250 mm)column, m obile phase was acetonitrile-water, detection wavelength was 210 nm, flow rate was 0.7 mL·min-1, column temperature was 30℃. The method showed repeatability and relative standard deviation was 0.15%, and the standard recoveries were 99.8%-100.2%. These quantitative results showed high accuracy.【期刊名称】《上海计量测试》【年(卷),期】2014(000)002【总页数】2页(P40-41)【关键词】高效液相色谱法;(S )-2-氨基丁酰胺盐酸盐【作者】郝玉红;朱力敏;薛韧婕【作者单位】上海市计量测试技术研究院;上海市计量测试技术研究院;上海市计量测试技术研究院【正文语种】中文(S)-2-氨基丁酰胺盐酸盐（简称ABAH），为白色或类白色固体粉末，主要用于抗癫痫、抗惊厥药物中间体。
酒石酸卡巴拉汀的产品说明书
参考文献：
[1]. Kurz A, Farlow M, Lefèvre G. Pharmacokinetics of a novel transdermal rivastigmine patch for the treatment of Alzheimer's disease: a review. Int J Clin Pract. 2009 May;63(5):799-805.
中文别名：
酒石酸卡巴拉汀;(S)-N-乙基-N-甲基氨基甲酸-3-[(1-二甲氨基)乙基]苯酯酒石酸盐;卡巴拉汀或利斯的明酒石酸盐;L-酒石酸卡巴拉汀;2,3',4-三溴二苯醚;N-乙基甲基氨基甲酰氯;Rivastigmine L-Tartrate L-酒石酸卡巴拉汀;酒石酸卡巴拉汀 USP标准品;酒石酸卡巴拉汀Rivastigmine tartrate;酒石酸卡巴拉汀标准品;酒石酸卡巴拉汀氢 EP标准品;酒石酸利凡斯的明;卡巴拉丁;卡巴拉汀酒石酸盐;重酒石酸卡巴拉汀;重酒石酸利凡斯的明;(S)-N-乙基-N-甲基氨基甲酸-3-[(S)-1-(二甲氨基)乙基]苯酯酒石酸盐;氘代重酒石酸利斯的明-d6;酒石酸卡巴拉汀 中文别名：卡巴拉汀或利斯的明酒石酸盐;酒石酸利伐斯的明;酒石酸利瓦斯汀;利斯的明酒石酸盐;卡巴拉汀重酒石酸盐
熔点
123-1250C
分子式
C18H28N2O8
分子量
400.423
闪点
145ºC
精确量
400.18457
PSA
147.84
LogP
0.6371
托马托花肽洒胶水产品说明说明书
SAFETY DATA SHEET1. IdentificationProduct identifierTomato Blossom Spray RTU Other means of identificationProduct code 32042Recommended use Agricutlural/ Horticultural Use- Foliar Fertilizer- Refer to product label Recommended restrictionsNone known.Manufacturer/Importer/Supplier/Distributor information Manufacturer Lawn and Garden Products, Inc.AddressPO Box 35000Company name Website Telephone Emergency Contact Number 1-559-994-9144Emergency phone numberCHEMTREC (24 hours):USA, Canada, Puerto Rico 1-800-424-3900E-mail Fresno, CA 937452. Hazard(s) identificationNot classified.Physical hazards Category 4Acute toxicity, oral Health hazardsCategory 2Skin corrosion/irritationCategory 2ASerious eye damage/eye irritationNot classified.Environmental hazards Not classified.OSHA defined hazardsLabel elementsSignal word WarningHazard statement Harmful if swallowed. Causes skin irritation. Causes serious eye irritation.Precautionary statementPreventionWash thoroughly after handling. Do not eat, drink or smoke when using this product. Wear protective gloves. Wear eye/face protection.ResponseIf swallowed: Call a poison center/doctor if you feel unwell. If on skin: Wash with plenty of water. If in eyes: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Specific treatment (see this label). Rinse mouth. If skin irritation occurs: Get medical advice/attention. If eye irritation persists: Get medical advice/attention. Take off contaminated clothing and wash before reuse.Storage Store away from incompatible materials.DisposalDispose of contents/container in accordance with local/regional/national/international regulations.Hazard(s) not otherwise classified (HNOC)None known.Supplemental information99.28% of the mixture consists of component(s) of unknown acute oral toxicity.3. Composition/information on ingredientsMixturesCAS number% Chemical name Common name and synonyms7664-38-2Phosphoric AcidOther components below reportable levels99.27692544710.7230745526*Designates that a specific chemical identity and/or percentage of composition has been withheld as a trade secret.4. First-aid measuresInhalation Move to fresh air. Call a physician if symptoms develop or persist.Skin contact Remove contaminated clothing. Wash with plenty of soap and water. If skin irritation occurs: Getmedical advice/attention. Wash contaminated clothing before reuse.Eye contact Immediately flush eyes with plenty of water for at least 15 minutes. Remove contact lenses, ifpresent and easy to do. Continue rinsing. Get medical attention if irritation develops and persists. Ingestion Rinse mouth. If vomiting occurs, keep head low so that stomach content doesn't get into the lungs.Get medical advice/attention if you feel unwell. Get medical attention if symptoms occur.Most importantsymptoms/effects, acute and delayed Symptoms may include stinging, tearing, redness, swelling, and blurred vision. May cause redness and pain. Severe eye irritation.Indication of immediate medical attention and special treatment needed Provide general supportive measures and treat symptomatically. Keep victim warm. Keep victim under observation. Symptoms may be delayed.General information Ensure that medical personnel are aware of the material(s) involved, and take precautions toprotect themselves. Show this safety data sheet to the doctor in attendance.5. Fire-fighting measuresSuitable extinguishing media Water fog. Foam. Dry chemical powder. Carbon dioxide (CO2).Unsuitable extinguishingmediaDo not use water jet as an extinguisher, as this will spread the fire.Specific hazards arising fromthe chemicalDuring fire, gases hazardous to health may be formed.Special protective equipmentand precautions for firefightersSelf-contained breathing apparatus and full protective clothing must be worn in case of fire.Fire-fightingequipment/instructionsMove containers from fire area if you can do so without risk.Specific methods Use standard firefighting procedures and consider the hazards of other involved materials. General fire hazards No unusual fire or explosion hazards noted.6. Accidental release measuresPersonal precautions, protective equipment and emergency procedures Keep unnecessary personnel away. Keep people away from and upwind of spill/leak. Wear appropriate protective equipment and clothing during clean-up. Do not touch damaged containers or spilled material unless wearing appropriate protective clothing. Ensure adequate ventilation. Local authorities should be advised if significant spillages cannot be contained. For personal protection, see section 8 of the SDS.Methods and materials for containment and cleaning up This product is miscible in water.Large Spills: Stop the flow of material, if this is without risk. Dike the spilled material, where this is possible. Cover with plastic sheet to prevent spreading. Absorb in vermiculite, dry sand or earth and place into containers. Following product recovery, flush area with water.Small Spills: Wipe up with absorbent material (e.g. cloth, fleece). Clean surface thoroughly to remove residual contamination.Never return spills to original containers for re-use. For waste disposal, see section 13 of the SDS.Environmental precautions Avoid discharge into drains, water courses or onto the ground.7. Handling and storagePrecautions for safe handling Do not taste or swallow. Avoid contact with eyes, skin, and clothing. Avoid contact with eyes. Avoidprolonged exposure. Provide adequate ventilation. Wear appropriate personal protectiveequipment. When using, do not eat, drink or smoke. Wash hands thoroughly after handling.Observe good industrial hygiene practices.Conditions for safe storage, including any incompatibilities Store in original tightly closed container. Keep container tightly closed. Store away from incompatible materials (see Section 10 of the SDS).8. Exposure controls/personal protectionOccupational exposure limitsUS. OSHA Table Z-1 Limits for Air Contaminants (29 CFR 1910.1000)Value Components TypePEL 1 mg/m3 Phosphoric Acid (CAS7664-38-2)US. ACGIH Threshold Limit ValuesValue Components TypeSTEL 3 mg/m3 Phosphoric Acid (CAS7664-38-2)TWA 1 mg/m3 US. NIOSH: Pocket Guide to Chemical HazardsValue Components TypeSTEL 3 mg/m3 Phosphoric Acid (CAS7664-38-2)TWA 1 mg/m3 Biological limit values No biological exposure limits noted for the ingredient(s).Appropriate engineering controls Good general ventilation (typically 10 air changes per hour) should be used. Ventilation rates should be matched to conditions. If applicable, use process enclosures, local exhaust ventilation, or other engineering controls to maintain airborne levels below recommended exposure limits. If exposure limits have not been established, maintain airborne levels to an acceptable level. Eye wash facilities and emergency shower must be available when handling this product.Individual protection measures, such as personal protective equipmentEye/face protection Face shield is recommended. Wear safety glasses with side shields (or goggles).Skin protectionHand protection Wear appropriate chemical resistant gloves.Other Wear appropriate chemical resistant clothing. Use of an impervious apron is recommended.Respiratory protection In case of insufficient ventilation, wear suitable respiratory equipment. Respiratory protection notrequired.Thermal hazards Wear appropriate thermal protective clothing, when necessary.General hygiene considerations Keep away from food and drink. Always observe good personal hygiene measures, such as washing after handling the material and before eating, drinking, and/or smoking. Routinely wash work clothing and protective equipment to remove contaminants.9. Physical and chemical properties Appearance Liquid.Physical state Liquid.Form Liquid.Color Colorless Odor Slight. Pungent Odor threshold Not available. pH 2.2Salt-Out / Crystallization Temp Not available. Melting point/freezing point Not available. Initial boiling point and boilingrangeNot available. Flash point Not available. Evaporation rate Not available. Flammability (solid, gas)Not available. Upper/lower flammability or explosive limits Flammability limit - lower(%)Not available.Flammability limit - upper(%)Not available.Explosive limit - lower (%)Not available.Explosive limit - upper (%)Not available.Vapor pressure 0.00001 hPa estimated Vapor density Not available.Relative density Not available.Solubility(ies)Solubility (water)Miscible Partition coefficient (n-octanol/water)Not available.Auto-ignition temperature Not available.Decomposition temperature Not available.ViscosityNot available.Other informationPercent volatile98.49 % estimated Pounds per gallon8.38 lb/gal typical10. Stability and reactivityReactivity The product is stable and non-reactive under normal conditions of use, storage and transport.Chemical stability Material is stable under normal conditions.Possibility of hazardous reactionsNo dangerous reaction known under conditions of normal use.Conditions to avoid Contact with incompatible materials.Incompatible materials Strong oxidizing agents.Hazardous decomposition productsNo hazardous decomposition products are known.11. Toxicological informationInformation on likely routes of exposureIngestionHarmful if swallowed.Inhalation Prolonged inhalation may be harmful.Skin contact Causes skin irritation.Eye contactCauses serious eye irritation.Symptoms related to thephysical, chemical andtoxicological characteristics Symptoms may include stinging, tearing, redness, swelling, and blurred vision. Skin irritation.Severe eye irritation. May cause redness and pain.Information on toxicological effectsAcute toxicity Harmful if swallowed. Not known.Test ResultsComponentsSpeciesPhosphoric Acid (CAS 7664-38-2)LD50Rabbit Dermal Acute 2740 mg/kg LD50RatOral 1530 mg/kg* Estimates for product may be based on additional component data not shown.Skin corrosion/irritation Causes skin irritation.Serious eye damage/eyeirritationCauses serious eye irritation.Respiratory or skin sensitizationRespiratory sensitizationNot available.Skin sensitizationThis product is not expected to cause skin sensitization.Germ cell mutagenicity No data available to indicate product or any components present at greater than 0.1% aremutagenic or genotoxic.Carcinogenicity This product is not considered to be a carcinogen by IARC, ACGIH, NTP, or OSHA.OSHA Specifically Regulated Substances (29 CFR 1910.1001-1050)Not listed.Reproductive toxicity This product is not expected to cause reproductive or developmental effects.Specific target organ toxicity -single exposureNot classified.Specific target organ toxicity -repeated exposureNot classified.Aspiration hazard Not available.Chronic effects Prolonged inhalation may be harmful.12. Ecological informationEcotoxicity The product is not classified as environmentally hazardous. However, this does not exclude thepossibility that large or frequent spills can have a harmful or damaging effect on the environment. Persistence and degradability No data is available on the degradability of this product.Bioaccumulative potential Not available.Mobility in soil No data available.Other adverse effects No other adverse environmental effects (e.g. ozone depletion, photochemical ozone creationpotential, endocrine disruption, global warming potential) are expected from this component. 13. Disposal considerationsDisposal instructions Collect and reclaim or dispose in sealed containers at licensed waste disposal site. Dispose ofcontents/container in accordance with local/regional/national/international regulations.Local disposal regulations Dispose in accordance with all applicable regulations.Hazardous waste code The waste code should be assigned in discussion between the user, the producer and the wastedisposal company.Waste from residues / unused products Dispose of in accordance with local regulations. Empty containers or liners may retain some product residues. This material and its container must be disposed of in a safe manner (see: Disposal instructions).Contaminated packaging Empty containers should be taken to an approved waste handling site for recycling or disposal.Since emptied containers may retain product residue, follow label warnings even after container isemptied.14. Transport informationDOTNot regulated as dangerous goods.IATANot regulated as dangerous goods.IMDGNot regulated as dangerous goods.15. Regulatory informationUS federal regulations This product is a "Hazardous Chemical" as defined by the OSHA Hazard CommunicationStandard, 29 CFR 1910.1200.All components are on the U.S. EPA TSCA Inventory List.This product is not known to be a "Hazardous Chemical" as defined by the OSHA HazardCommunication Standard, 29 CFR 1910.1200.TSCA Section 12(b) Export Notification (40 CFR 707, Subpt. D)Not regulated.CERCLA Hazardous Substance List (40 CFR 302.4)Phosphoric Acid (CAS 7664-38-2)Listed.SARA 304 Emergency release notificationNot regulated.OSHA Specifically Regulated Substances (29 CFR 1910.1001-1050)Not listed.Superfund Amendments and Reauthorization Act of 1986 (SARA)Hazard categories Immediate Hazard - YesDelayed Hazard - NoFire Hazard - NoPressure Hazard - NoReactivity Hazard - NoSARA 302 Extremely hazardous substanceNot listed.NoSARA 311/312 HazardouschemicalSARA 313 (TRI reporting)Not regulated.Other federal regulationsClean Air Act (CAA) Section 112 Hazardous Air Pollutants (HAPs) ListNot regulated.Clean Air Act (CAA) Section 112(r) Accidental Release Prevention (40 CFR 68.130)Not regulated.Not regulated.Safe Drinking Water Act(SDWA)US state regulationsUS. Massachusetts RTK - Substance ListPhosphoric Acid (CAS 7664-38-2)US. New Jersey Worker and Community Right-to-Know ActPhosphoric Acid (CAS 7664-38-2)US. Pennsylvania Worker and Community Right-to-Know LawPhosphoric Acid (CAS 7664-38-2)US. Rhode Island RTKPhosphoric Acid (CAS 7664-38-2)US. California Proposition 65WARNING: This product contains a chemical known to the State of California to cause cancer and birth defects or otherreproductive harm.International InventoriesCountry(s) or region Inventory name On inventory (yes/no)* Australia Australian Inventory of Chemical Substances (AICS)Yes Canada Domestic Substances List (DSL)Yes Canada Non-Domestic Substances List (NDSL)No China Inventory of Existing Chemical Substances in China (IECSC)Yes Europe European Inventory of Existing Commercial ChemicalYesSubstances (EINECS)Europe European List of Notified Chemical Substances (ELINCS)No Japan Inventory of Existing and New Chemical Substances (ENCS)No Korea Existing Chemicals List (ECL)No New Zealand New Zealand InventoryYes Philippines Philippine Inventory of Chemicals and Chemical SubstancesNo(PICCS)United States & Puerto Rico Toxic Substances Control Act (TSCA) InventoryYes *A "Yes" indicates that all components of this product comply with the inventory requirements administered by the governing country(s)A "No" indicates that one or more components of the product are not listed or exempt from listing on the inventory administered by the governingcountry(s).16. Other information, including date of preparation or last revisionIssue date07-21-2015Revision date07-22-2015Version #04Disclaimer Lawn and Garden Products cannot anticipate all conditions under which this information and itsproduct, or the products of other manufacturers in combination with its product, may be used. It isthe user’s responsibility to ensure safe conditions for handling, storage and disposal of theproduct, and to assume liability for loss, injury, damage or expense due to improper use. While theinformation contained herein are presented in good faith and believed to be accurate, it is providedfor your guidance only. Because many factors may affect processing or application, werecommend that you make tests to determine the suitability of a product for your particular purposeprior to use. No warranties of any kind, either expressed or implied, including warranties ofmerchantability or fitness for a particular purpose, are made regarding products described orinformation set forth, or that the products, or information may be used without infringing theintellectual property rights of others. In no case shall the information provided be considered apart of our terms and conditions of sale. Further, you expressly understand and agree that theinformation furnished by our company hereunder are given gratis and we assume no obligation orliability for the information given or results obtained, all such being given and accepted at your risk.。
噻托溴铵reaxys_路线总结
Query1. QueryView in Reaxys87.7 %13:Tiotropium bromide31.5 g (0.1 mol) methylscopinium hexafluorophosphate (Example 9) and 30.5 g (0.10 mol) of 2,2'-methyl dithienylgly-colate are dissolved in 400 ml acetone and stirred in the presence of 90 g of zeolite of type 4A (Na12Al12Si12O48.x.nH2O) and 0.2 g (1 mmol) potassium-tert.-butoxide over a period of 20-24 hours at 0° C.The reaction mixture is filtered,the filtrate is combined with a solution of 8.7 g of LiBr (8.7 g 0.10 mol in 100 ml acetone).The product that crystallisesout is separated off by filtration, washed with acetone and then dried.A yield of 41.4 g (87.7percent) is obtained, witha 90percent conversion level.Stage 1:With potassium tert-butylate in acetone, Time= 20 - 24h, T= 0 °C , Molecular sieveStage 2:With lithium bromide in acetone, Product distribution / selectivityPatent; Brandenburg, Joerg; US2010/63289; (2010); (A1) EnglishView in Reaxys87.7 %6:EXAMPLE 6Tiotropium bromide31.5 g (0.1 mol) methylscopinium hexafluorophosphate (Example 1) and 30.5 g (0.10 mol) of 2,2'-methyl dithienylgly-colate are dissolved in 400 ml acetone and stirred in the presence of 90 g of zeolite of type 4 Å (Na12Al12Si12O48*nH2O) and 0.2 g (1 mmol) potassium-tert.-butoxide over a period of 20-24 hours at 0° C.The reaction mixture is filtered, the filtrate is combined with a solution of 8.7 g of LiBr (8.7 g 0.10 mol in 100 ml acetone).The product that crystallises out is separated off by filtration, washed with acetone and then dried.A yield of 41.4 g (87.7percent) is obtained, with a 90percent conversion level.Stage 1:With potassium tert-butylate, zeolite Na12Al12Si12O48*nH2O 4 Å in acetone, T= 0 °CStage 2:With lithium bromide in acetone, Product distribution / selectivityPatent; BOEHRINGER INGELHEIM PHARMA GMBH and CO. KG; US2010/105898; (2010); (A1) EnglishView in Reaxys48 %5:Example 5 Tiotropium Bromide 1.6 g (5 mmol) methylscopinium hexafluorophosphate (Example 1) and 1.25 g (5mmol) methyl dithienylglycolate are stirred in 50 ml acetone and in the presence of 2 g powdered molecular sieve 4A(Fluka) and 6 mg potassium-tert.-butoxide at 0° C. for 4 h. The reaction mixture is filtered, washed with 20 ml acetone,the filtrate is combined stepwise with a solution of 0.7 g LiBr (13 mmol) in 11 ml acetone. The unreacted material thatcrystallises out is separated off by filtration (fractionated precipitation). The crystal fractions were filtered off and dried.The composition of the fractions was determined by thin layer chromatography. The tiotropium bromide fractions weresuction filtered, washed with acetone, recrystallised from water, washed with acetone and dried. 1.2 g (48percent yieldbased on the compound according to Example 1 used). Tiotropium bromide was isolated in this way. Purity HPLC:99.8percent, TLC: no visible contaminationStage 1:With potassium tert-butylate in acetone, Time= 4h, T= 0 °C , Molecular sieveStage 2:With lithium bromide in acetone, Product distribution / selectivityPatent; Boehringer Ingelheim Pharma GmbH and Co. KG; US2007/27320; (2007); (A1) EnglishView in Reaxys35 %4:Example 2 Tiotropium Bromide 1.6 g (5 mmol) methylscopinium hexafluorophosphate (Example 1) and 2.0 g (7.8mmol) methyl dithienylglycolate are refluxed in 50 ml acetone and in the presence of 10 g molecular sieve 4A for 50-70hours. The reaction mixture is filtered, the filtrate is combined with a solution of 0.3 g of LiBr in 10 ml acetone. The stillunreacted N-methylscopinium bromide that crystallises out is separated off by filtration. After the addition of another0.6 g LiBr (dissolved in acetone) tiotropium bromide is precipitated in an isolated yield of 30percent (based on thecompound of Example 1 used).Stage 1:With 4-(N,N-dimethlyamino)pyridine in acetone, Time= 24h, Molecular sieve, Heating / refluxStage 2:With lithium bromide in acetone, Product distribution / selectivityPatent; Boehringer Ingelheim Pharma GmbH and Co. KG; US2007/27320; (2007); (A1) EnglishView in Reaxys30 %2:Example 2 Tiotropium Bromide 1.6 g (5 mmol) methylscopinium hexafluorophosphate (Example 1) and 2.0 g (7.8mmol) methyl dithienylglycolate are refluxed in 50 ml acetone and in the presence of 10 g molecular sieve 4A for 50-70hours. The reaction mixture is filtered, the filtrate is combined with a solution of 0.3 g of LiBr in 10 ml acetone. The stillunreacted N-methylscopinium bromide that crystallises out is separated off by filtration. After the addition of another0.6 g LiBr (dissolved in acetone) tiotropium bromide is precipitated in an isolated yield of 30percent (based on the compound of Example 1 used).Stage 1:in acetone, Time= 50 - 70h, Heating / reflux, Molecular sieveStage 2:With lithium bromide in acetone, Product distribution / selectivityPatent; Boehringer Ingelheim Pharma GmbH and Co. KG; US2007/27320; (2007); (A1) EnglishView in Reaxystropium bromide obtained was found to have the XRPD,DSC and TGA spectra shown in Figures 1, 2 and 3 respec-tively.Molar yield = 97.76percentHPLC purity = 99.83percentin dichloromethane, acetonitrile, Time= 30.3333h, T= 25 - 30 °CPatent; GENERICS [UK] LIMITED; MYLAN INDIA PRIVATE LIMITED; GORE, Vinayak Govind; MANOJKUMAR,Bindu; SHINDE, Dattatraya; KOKANE, Dattatrey; WO2011/15883; (2011); (A1) EnglishView in Reaxys97.76 %Tiotropium Bromide (1)The purified tiotropium base (3) (1 eq) was dissolved in DCM (10 vol) and acetonitrile (3 vol)and purged with methyl bromide gas for 20 minutes. The solution was kept at 25-300C for 30 hours. The precipitatedsolid was filtered and washed with DCM (20 vol). Drying of the solid at 25-300C under vacuum gave the product,tiotropium bromide (1), as a white solid.Molar yield = 97.76percentHPLC purity = 99.83percent1H NMR (300 MHz,CD3OD): 7.45 (2H dd), 7.23 (2H dd), 7.00 (2H dd), 5.27 (IH t), 4.60 (IH br s OH), 3.35 (8H m), 3.10 (2H s), 2.85 (2Hdt), 2.10 (2H d).MS: 392.3 (M+l)in dichloromethane, acetonitrile, Time= 30.3333h, T= 20 - 30 °CPatent; GENERICS [UK] LIMITED; MYLAN INDIA PRIVATE LIMITED; GORE, Vinayak Govind; MANOJKUMAR,Bindu; SHINDE, Dattatraya; KOKANE, Dattatrey; WO2011/15884; (2011); (A1) EnglishView in Reaxys97.2 %11:Example 11: Synthesis of tiotropium bromideExample 11: Synthesis of tiotropium bromide A solution of 22percent methyl bromide in acetone was prepared byscrubbing gas into a dark glass bottle containing 1.6 kg of acetone and placed on scales until obtaining 1.95 kg ofmethyl bromide solution in acetone.In a reaction flask were charged, in this order:scopine ester (3): 242.6g (0.6426moles, 1 eq) DMF: 388 mL,acetone: 1370 mL(acetone/DMF ratio: about 3.5:1)The suspension, maintained under me-chanical stirring, was heated to 40±5°C until obtaining complete dissolution of the solid.The clear (pale yellow colored)solution was cooled to 20°C and, keeping the flask immersed in a water bath, a 22.1percent (w/w) solution of methylbromide in acetone: 636 mL, equal to about 1.8 eq. of MeBr, was rapidly dropped, by addition funnel, on the clearsolution.In a few hours of stirring at room temperature, a progressive precipitation of white solid was observed.Thereaction mixture was maintained under stirring at room temperature. after about 4 days (90-100 h) a sampling wascarried out for reaction control.The reaction mixture appeared as a heterogeneous suspension made up of clear motherwaters and grainy and heavy solid, which tended to rapidly settle on the flask bottom.The solid was filtered, and flaskand solid were washed over a filter with: 863 mL of acetone/DMF mixture (5:1), 431 mL of acetone/DMF mixture (5:1),2x431 mL of acetone. The solid thus obtained was dried over the filter under Nitrogen current for 2-3h. At the end ofthe drying the solid appeared white and powdery.Crude dry Tiotropium bromide weight: 295.1 g (yield=97.2percent) .in N,N-dimethyl-formamide, acetone, T= 20 °CPatent; LUSOCHIMICA S.P.A.; CIPOLLONE, Amalia; FATTORI, Daniela; FINCHAM, Christofer Ingo;WO2013/46138; (2013); (A1) EnglishView in Reaxys97.3%2:preparation of crude tiotropium bromideN-demethyltiotropium (66 g; 0.17 mol) was dissolved in dimethylformamide (330 ml) and the solution was cooled to atemperature between 0 °C and 5 °C. A solution of bromomethane in 2-methyltetrahydrofuran (132 ml; 0.72 mol) wasadded and the reaction mixture stirred overnight at 0 °C - 5 °C. The content of N-demethyltiotropium in the reactionmixture was 3.6percent by HPLC. Then the reaction mixture was heated up to a temperature between 10 °C and 15°C and stirred at this temperature range over 2 hours. The content of N-demethyltiotropium in the reaction mixturedecreased to 1.7percent. The reaction mixture was heated to a temperature between 25 °C and 30 °C and stirred atthat temperature range over 1 hour. The content of N- demethyltiotropium in the reaction mixture decreased to 1.0per-cent by HPLC. 2- Methyltetrahydrofuran (594 ml) was added to the reaction mixture previously cooled to 0 °C - 5 °C,the suspension was stirred over 1 hour while maintaining the temperature between 0 °C and 5 °C, the product wasfiltered and washed with 2-methyltetrahydrofuran (297 ml) previously cooled to a temperature between.0 °C and 5 °C.The purity of the wet product was 99.48percent and the content of N-demethyltiotropium was 0.33percent (by HPLC).The wet product was re-s urried in dimethylformamide (297 ml) over 1 hour, was filtered, was washed with 2- methyl-tetrahydrofuran (297 ml) previously cooled' to 0 °C - 5 °C and dried. Crude tiotropium bromide (804 g; 97.3percent ofthe theoretical yield) was obtained with a purity of .99.77percent and with a residual content of N-demethyltiotropiumof 0.16percent (by HPLC) . -in 2-methyl tetrahydrofuran, N,N-dimethyl-formamide, T= 0 - 30 °CPatent; HOVIONE INTERNATIONAL LTD; KING, Lawrence; SOBRAL, Luis; TEMTEM Marcio; ANTUNES, Rafael;NUNES, Bruna; WO2013/117886; (2013); (A1) EnglishView in Reaxys95 %Preparation of Anhydrous Tiotropium Bromide from Scopine di-(2-thienyl)glycolatePreparation of Anhydrous Tiotropium Bromide from Scopine di-(2-thienyl)glycolateScopine di-(2-thienyl)glycolate (1 eq) was dissolved in acetone (35 vol) at 25-30° C. and methyl bromide in acetonitrile(50percent w/w solution, 5 vol) was added.The mixture was stirred at 25-30° C. for 24 hours and the precipitated solid was filtered and washed with acetone (5vol).The solid was dried at 60° C. under vacuum to afford the product as a white solid.The crude anhydrous tiotropium bromide obtained was found to have the XRPD, DSC and TGA spectra shown in , 2and 3 respectively.Molar yield=95percentHPLC purity=99.5-99.7percentPurification of Anhydrous Tiotropium Bromidein acetone, acetonitrile, Time= 24h, T= 25 - 30 °CPatent; GENERICE (UK) LIMITED; US2012/149725; (2012); (A1) EnglishView in Reaxys88 % 2.c:c) Preparation of the Tiotropium BromideMethyl bromide (5.1 kg) is piped into the scopine ester solution obtained by the method described above at 20° C. Thecontents of the apparatus are stirred at 30° C. for about 2.5 days. 70 L of DMF are distilled off at 50° C. in vacuo. Thesolution is transferred into a smaller apparatus. It is rinsed with DMF (10 L). Additional DMF is distilled off at 50° C. invacuo until a total amount of distillate of about 100 L is obtained. This is cooled to 15° C. and stirred for 2 hours at thistemperature. The product is isolated using a suction filter drier, washed with 15° C. cold DMF (10 L) and 15° C. coldacetone (25 L). It is dried at max. 50° C. for max. 36 hours in a nitrogen current. Yield: 13.2 kg (88percent); [0060]Melting point: 200-230° C. (depending on the purity of the starting product); [0061] The crude product thus obtained(10.3 kg) is added to methanol (66 L). The mixture is refluxed to dissolve it. The solution is cooled to 7° C. and stirredfor 1.5 h at this temperature. The product is isolated using a suction filter drier, washed with 7° C. cold methanol (11L) and dried for max. 36 h at about 50° C. in a nitrogen current. [0062] Yield: 9.9 kg (96percent); [0063] Melting point:228° C. (determined by TLC at a heating rate of 10 K/min). [0064] If desired, the product thus obtained may be convertedin the crystalline monohydrate of tiotropium bromide. The following method may be used. [0065] 15.0 kg of tiotropiumbromide are added to 25.7 kg of water in a suitable reaction vessel. The mixture is heated to 80-90° C. and stirred atconstant temperature until a clear solution is formed. Activated charcoal (0.8 kg), moistened with water, is suspendedin 4.4 kg of water, this mixture is added to the solution containing tiotropium bromide and rinsed with 4.3 kg of water.The mixture thus obtained is stirred for at least 15 min. at 80-90° C. and then filtered through a heated filter into anapparatus which has been preheated to an outer temperature of 70° C. The filter is rinsed with 8.6 kg of water. Thecontents of the apparatus are cooled to a temperature of 20-25° C. at a rate of 3-5° C. every 20 minutes. Using coldwater the apparatus is cooled further to 10-15° C. and crystallisation is completed by stirring for at least another hour.The crystals are isolated using a suction filter drier, the crystal slurry isolated is washed with 9 L of cold water (10- 15°C.) and cold acetone (10-15° C.). The crystals obtained are dried at 25° C. for 2 hours in a nitrogen current. [0066]Yield: 13.4 kg of tiotropium bromide monohydrate (86percent of theory). [0067] Melting point: 230° C. (determined byTLC at a heating rate of 10 K/min).in DMF (N,N-dimethyl-formamide), Time= 60h, T= 20 - 30 °CPatent; Boehringer Ingelheim Pharma GmbH and Co. KG; US2003/236409; (2003); (A1) EnglishView in Reaxys2; 3:Example 2: Preparation of crude Tiotropium bromide <n="29"/>[0109] 0.52 g of N-demethyl tiotropium (1.39 mmol)was suspended in 5.23 iriL of CH3CN under nitrogen.[0110] 1.35 g of CH3Br 50percent w/w solution in CH3CN (0.0071mol) were loaded, and the suspension was left under stirring at 220C for 12 hours. The product was filtered and washedwith ImI of CH3CN.[0111] 572 mg of wet Tiotropium bromide were obtained (HPLC purity 99.89percent, dithienylglycolicacid not detected).; Example 3 : Preparation of Tiotropium bromide[0112] 4.96g of N-demethyl tiotropium (13.2 mmol)were loaded in a flask under nitrogen with 49.6mL of CH3-CN. A suspension was obtained. 12.61g of CH3Br 50percentw/w -CH3CN solution- (0.066 mol) were loaded.[0113] The suspension was left under stirring at 22°C for 64 hours. Theproduct was filtered and washed with 2mL of CH3CN. [0114] 6.93g of wet Tiotropium were obtained, and dried undervacuum at 45°C for 22h (residual pressure 4 mbar) . 5.9 g of dry product (purity 99.8percent, dithienylglycolic acid-notdetected) were obtained.in acetonitrile, Time= 12 - 64h, T= 22 °C , Product distribution / selectivityPatent; SICOR INC.; WO2007/75838; (2007); (A2) EnglishView in ReaxysPreparation of anhydrous tiotropium bromide from scopine di-(2-thienyl)glycolateScopine di-(2-thienyl)glycolate (1 eq)was dissolved in acetone (35 vol) at 25-30°C and methyl bromide in acetonitrile (50percent w/w solution, 5 vol) wasadded. The mixture was stirred at 25-3O0C for 24 hours and the precipitated solid was filtered and washed with acetone(5 vol). The solid was dried at 60°C under vacuum to afford the product as a white solid. The crude anhydrous tiotropiumbromide obtained was found to have the XRPD, DSC and TGA spectra shown in Figures 1, 2 and 3 respectively.Molaryield = 95percentHPLC purity = 99.5-99.7percentPurification of anhydrous tiotropium bromideCrude anhydrous tio-teopium bromide (1 eq) was taken in DMSO (2 vol) and stirred for 1 hour at 25-3O0C. Acetone (25 vol) was slowlyadded and the mixture was chilled to 0-5°C and stirred at 0-5°C for 30 minutes. The solid was filtered and washed withacetone (3 vol) and dried under vacuum at 600C for 12 hours. The purified anhydrous tiotropium bromide obtained wasfound to have the XRPD, DSC and TGA spectra shown in Figures 1, 2 and 3 respectively.Molar yield = 98percentHPLCpurity > 99.9percentThe crude and purified samples of anhydrous tiotropium bromide prepared in the above exampleswere found to be substantially pure polymorphically with no levels of other- forms detected (>99.7percent polymorph-ically pure, as measured by XRPD). The purified anhydrous tiotropium bromide prepared was also found to be verystable chemically and polymorphically with no conversion over time to other polymorphs. The stability of the samplewas tested by subjecting the sample to accelerated stability conditions (400C + 2°C temperature and 75percent +/-5percent relative humidity) for 6 months. The chemical purity (measured by related substances and purity assays byHPLC) and polymorphic purity (measured by XRPD, DSC and TGA) were monitored for 6 months and the sample wasfound to be chemically and polymorphically stable even after 6 months under accelerated stability conditions.TheXRPDs were recorded on a Bruker D8 Advance Diffractometer, using Cu Kαl radiation as the X-ray source and LynxEyeas the detector, with a 2ψ range of from 3° to 50°, a step-size of 0.05° and a time/step of lsec.The DSCs were recordedon a Perkin Elmer Pyris 6 Instrument over a temperature range of from 250C to 25O0C at a rate of heating of 10°C/min.The TGAs were recorded on a Perkin Elmer Pyris 1 Instrument over a temperature range of from 250C to 250°C at arate of heating of 10°C/min.in acetone, acetonitrile, Time= 24h, T= 25 - 30 °CPatent; GENERICS [UK] LIMITED; MYLAN INDIA PRIVATE LIMITED; GORE, Vinayak Govind; MANOJKUMAR,Bindu; SHINDE, Dattatraya; KOKANE, Dattatrey; WO2011/15882; (2011); (A2) EnglishView in Reaxys2.65 g2:Example 22.0 g of scopine di(2-thienyl)glycolate (5.3 mmol) was dissolved in 20 ml of a mixture of dichloromethane (8 ml) andacetonitrile (12 ml) at 55°C. The solution was cooled down to 33°C and 5.46 g of 55percent MeBr in acetonitrile (5.1equivalents) was added. The solution was stirred and left to cool freely without stirring still for 48 hours. The formedcrystalline product was filtered, washed with 30 ml of dichloromethane, and dried in a vacuum oven at 35°C for 6 hours.2.65 g of white crystals were obtained,HPLC purity 99.60percent. The content of solvents (determined by GC): di-chloromethane 69,000 ppm, acetonitrile 8,200 ppm.in dichloromethane, acetonitrile, Time= 48h, T= 20 °C , Concentration, TimePatent; ZENTIVA K.S.; CERNA, Igor; RIDVAN, Ludek; KRAL, Vladimir; HAJICEK, Josef; DAMMER, Ondrej;WO2013/79040; (2013); (A1) EnglishView in Reaxys30.8 g3:Example 3 (Reference example according to the method of patent EP0418716)23.6 g of the scopine ester I (39.7 mmol) was dissolved in 230 ml of a mixture of dry dichloromethane (90 ml) and dryacetonitrile (140 ml) at 55°C, then the solution was cooled to 33°C and 54.0 g of 55percent MeBr in dry acetonitrile (5.0equivalents) were added. The solution was left to cool freely and then it was stirred at the room temperature for 19hours. The resulting crystalline product was filtered, washed with 900 ml of dichloromethane and dried in a vacuumdrier (5 kPa) at 35°C for 17 hours. 30.8 g of white crystals were obtained, HPLC purity 99.74percent. The content ofsolvents (measured with gas chromatography): dichloromethane 67000 ppm, acetonitrile 4300 ppm.in dichloromethane, acetonitrile, Time= 19h, T= 20 - 33 °CPatent; ZENTIVA, K.S.; CERNA, Igor; HAJICEK Josef; WO2013/135219; (2013); (A1) EnglishView in Reaxys30.8 g7; 9:Quaternization of the scopine ester I with methyl bromideExample 9 (Reference example reproducing the method of patent EP0418716) 23.6 g of the scopine ester I (39.7 mmol)was dissolved in 230 ml of a mixture of dry dichloromethane (90 ml) and dry acetonitrile (140 ml) at 55°C, then thesolution was cooled to 33°C and 54.0 g of 55percent MeBr in dry acetonitrile (5.0 equivalents) were added. The solutionwas left to cool down freely, and subsequently stirred at the room temperature for 19 hours. The resulting crystallineproduct was filtered, washed with 900 ml of dichloromethane and dried in a vacuum drier (5 kPa) at 35 °C for 17 hours.30.8 g of white crystals were obtained, UPLC purity 99.74percent. Solvent content (determined by gas chromatogra-phy): dichloromethane 67000 ppm, acetonitrile 4300 ppm.in dichloromethane, acetonitrile, Time= 19h, T= 20 - 33 °CPatent; ZENTIVA, K.S.; CERNA, Igor; HAJICEK, Josef; WO2013/143510; (2013); (A1) EnglishView in Reaxys15.51 g3:Preparation of Tiotropium Bromide (1) in NMPExample 3Preparation of Tiotropium Bromide (1) in NMPTo a solution of 13.2 g (39.1 mmol) desmethyl-tiotropium (4) in 30 mL NMP, 16.5 mL (115 mmol, 2.93 eq.) of a 1:1 (w/w) solution of methyl bromide in NMP were added.The mixture was stirred overnight at room temperature, whereupon a dense suspension formed.After addition of 20 mL acetonitrile, the suspension was filtered, washed with 20 mL acetonitrile, and dried with highvacuum overnight at 30° C., yielding 15.51 g of off-white powder.Residual solvents were detected by GC analysis.The XRPD pattern complied with the one shown in .1H-NMR (300 MHz, d-DMSO): 7.52 (dd, J=5.0 Hz, 1.1, 2H), 7.41 (s, 1H), 7.13 (dd, J=3.6, 1.1 Hz, 2H), 7.01 (dd, J=5.0,63.7 Hz, 2H), 5.12 (t, J=5.8 Hz, 1H),4.13 (bd, J=5.8 Hz, 2H), 3.50 (s, 2H), 3.25 (s, 3H), 3.05 (s, 3H), 2.8-2.6 (m, 2H),1.93 (s, 1H), 1.87 (s, 1H).13C-NMR (75.5 MHz, d-DMSO): 170.2, 147.1, 127.3, 126.7, 126.3, 76.8, 65.0, 64.2, 56.5, 54.1, 47.6, 28.7.6in 1-methyl-pyrrolidin-2-one, T= 20 °CPatent; CERBIOS-PHARMA SA; MEREU, Andrea; MOROSOLI, Moreno; PENNE', Umberto; PERSEGHINI,Mauro; US2014/303373; (2014); (A1) EnglishView in Reaxys(37.5 mmol) scopine methobromide are added and a solution of 2.59 g (38 mmol) imidazole and 1.52 g (38 mmol)sodium hydride (60percent) in 30 ml dimethylacetamide is added dropwise at 20° C. and the mixture is stirred for 1 hat 20° C. After cooling to -4° C. 50 ml of a 33percent solution of hydrogen bromide in glacial acetic acid are addeddropwise while the temperature does not exceed 20° C. Then 50 ml methanol are added and the mixture is stirred for3 h at 20° C. The reaction mixture is extracted with 500 ml toluene and, after separation of the toluene phase, crystallisedfrom 150 ml isopropanol at 0° C. The crude product is filtered off, washed with 30 ml cold isopropanol and dried invacuo. Yield 14.1 g (80percent, based on scopine methobromide).Stage 1:With chloro-trimethyl-silane, 1,1'-carbonyldiimidazole in isobutyramide, Time= 0.5h, T= 20 °CStage 2:With 1H-imidazole, sodium hydride in isobutyramide, Time= 1h, T= 20 °CStage 3:With methanol, hydrogen bromide in isobutyramide, acetic acid, Time= 3h, T= -4 - 20 °C , Product distribution /selectivityPatent; Boehringer Ingelheim Pharma GmbH and Co., KG; US2006/47120; (2006); (A1) EnglishView in Reaxys48 %9:17.9 g (165 mmol) chlorotrimethylsilane are added dropwise at 0° C. to a solution of 39.3 g (150 mmol) sodiumdithienylglycolate in 117 ml tetrahydrofuran. After 60 min stirring at 10-20° C. the mixture is cooled to 0° C. and a solutionof 24.3 g (150 mmol) carbonyldiimidazole in 105 ml dimethylformamide is added dropwise. After a further 30 min stirring30.3 g (121 mmol) scopine methobromide are added and the mixture is stirred for a further 60 min at 10-20° C. It iscooled to 10° C. and a solution of 16.8 g (150 mmol) potassium tert. butoxide in 90 ml tetrahydrofuran is added dropwiseat 10-20° C. and the mixture is stirred for 60 min at 20° C. After cooling to 0° C. 60 ml 62percent hydrobromic acid areadded dropwise while the temperature does not exceed 20° C. After 40 min stirring the reaction mixture is stirred into1150 ml isopropanol at 20° C. and cooled to 10° C. The crude product is filtered off, washed with 70 ml cold isopropanoland dried in vacuo. Yield 61.5 g reddish-brown crystals, TLC corresponds to comparison. The crude product is dissolvedin 615 ml methanol with 6.15 g activated charcoal at reflux temperature and filtered. Then 570 ml methanol are distilledoff and the solution is cooled to 10° C. The crystals are filtered off, washed with 35 ml cold methanol and dried. Yield40.9 g whitish-beige crystals, TLC corresponds to comparison. The crystals thus obtained are dissolved in 94 ml waterwith 2.2 g activated charcoal at 80° C. and filtered, and then washed with 24 ml water. After cooling to 15° C. thetiotropium bromide monohydrate which has crystallised out is filtered off, washed with 25 ml water and 35 ml acetoneand dried. Yield 28.6 g (48percent based on the scopine methobromide used).Stage 1:With chloro-trimethyl-silane, 1,1'-carbonyldiimidazole in tetrahydrofuran, DMF (N,N-dimethyl-formamide),Time= 1.5h, T= 0 - 20 °CStage 2:in tetrahydrofuran, DMF (N,N-dimethyl-formamide), Time= 1h, T= 10 - 20 °CStage 3:With potassium tert-butylate, hydrogen bromide, Product distribution / selectivity, more than 3 stagesPatent; Boehringer Ingelheim Pharma GmbH and Co., KG; US2006/47120; (2006); (A1) EnglishView in Reaxys48 %8:5.43 g (50 mmol) chlorotrimethylsilane are added dropwise at 20-30° C. to a solution of 13.1 g (50 mmol) sodiumdithienylglycolate in 25 ml tetrahydrofuran. After 60 min stirring 8.1 g (50 mmol) carbonyldiimidazole and after another30 min 10.01 g (40 mmol) scopine methobromide are added and the mixture is stirred for a further 30 min. Then asolution of 2.60 g (38 mmol) imidazole and 1.65 g (38 mmol) sodium hydride (55percent) in 25 ml dimethylformamideis added dropwise at 20° C. and the mixture is stirred for 1 h at 20° C. After cooling to 0° C. 20 ml 62percent hydrobromicacid are added dropwise while the temperature does not exceed 20° C. After 40 min stirring the reaction mixture isstirred into 350 ml isopropanol at 20° C. and cooled to 10° C. The crude product is filtered off, washed with 50 ml coldisopropanol and dried in vacuo. Yield 18.9 g reddish-brown crystals, TLC corresponds to the comparison. The crudeproduct is dissolved in 100 ml methanol with 2.2 g activated charcoal at reflux temperature and filtered. Then the solutionis evaporated down to 30 ml and cooled to 3° C. The crystals are filtered off, washed with 5 ml cold methanol and dried.Yield 12.1 g whitish-beige crystals, TLC corresponds to the comparison. The crystals thus obtained are dissolved in28 ml water with 1.2 g activated charcoal at 80° C. and filtered. After cooling to 15° C. the tiotropium bromide mono-hydrate which has crystallised out is filtered off and dried. Yield 9.4 g (48percent based on the scopine methobromideused).Stage 1:With chloro-trimethyl-silane, 1,1'-carbonyldiimidazole in tetrahydrofuran, Time= 1.5h, T= 20 - 30 °CStage 2:With 1H-imidazole, sodium hydride in tetrahydrofuran, DMF (N,N-dimethyl-formamide), Time= 1.5h, T= 20°CStage 3:With hydrogen bromide in tetrahydrofuran, DMF (N,N-dimethyl-formamide), isopropyl alcohol, Time=0.666667h, T= 0 - 20 °C , Product distribution / selectivityPatent; Boehringer Ingelheim Pharma GmbH and Co., KG; US2006/47120; (2006); (A1) EnglishView in Reaxys。
大肠杆菌高产L丙氨酸
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGYProduction of L-alanine by metabolically engineered Escherichia coliXueli Zhang&Kaemwich Jantama&J.C.Moore&K.T.Shanmugam&L.O.IngramReceived:23May2007/Revised:13August2007/Accepted:16August2007/Published online:15September2007 #Springer-Verlag2007Abstract Escherichia coli W was genetically engineered to produce L-alanine as the primary fermentation product from sugars by replacing the native D-lactate dehydroge-nase of E.coli SZ194with alanine dehydrogenase from Geobacillus stearothermophilus.As a result,the heterolo-gous alanine dehydrogenase gene was integrated under the regulation of the native D-lactate dehydrogenase(ldhA) promoter.This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation.Strain XZ111accumulated alanine as the primary product during glucose fermentation.The methyl-glyoxal synthase gene(mgsA)was deleted to eliminate low levels of lactate and improve growth,and the catabolic alanine racemase gene(dadX)was deleted to minimize conversion of L-alanine to D-alanine.In these strains,re-duced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth.This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production.The resulting strain, XZ132,produced1,279mmol alanine from120g l−1 glucose within48h during batch fermentation in the mineral salts medium.The alanine yield was95%on a weight basis(g g−1glucose)with a chiral purity greater than99.5%L-alanine.Keywords Alanine.Fermentation.E.coli.Evolution. GlycolysisIntroductionWorldwide production of L-alanine has been estimated at 500tons per year(Ikeda2003).In pharmaceutical and veterinary applications,L-alanine is used with other L-amino acids as a pre-and postoperative nutrition therapy(Hols et al.1999).Alanine is also used as a food additive because of its sweet taste(Lee et al.2004).The use of L-alanine is limited in part by the current high cost.L-Alanine is pro-duced commercially by the enzymatic decarboxylation of L-aspartic acid using immobilized cells or cell suspensions of Pseudomonas dacunhae as a biocatalyst with a yield greater than90%(Shibatani et al.1979).The substrate for this enzymatic production process,L-aspartate,is usually pro-duced from fumarate by enzymatic catalysis with aspartate ammonia-lyase.Fumaric acid is produced primarily from petroleum,a nonrenewable feedstock.An efficient fermen-tative process with a renewable feedstock such as glucose offers the potential to reduce L-alanine cost and facilitate a broad expansion of the alanine market into other products.Alanine is a central intermediate(Fig.1)and an essential component of cellular proteins.Most microorganisms produce alanine only for biosynthesis using a glutamate–pyruvate transaminase(Hashimoto and Katsumata1998). Some organisms such as Arthrobacter oxydans(Hashimoto and Katsumata1993;Hashimoto and Katsumata1998; Hashimoto and Katsumata1999),Bacillus sphaericus (Ohashima and Soda1979),and Clostridium sp.P2Appl Microbiol Biotechnol(2007)77:355–366DOI10.1007/s00253-007-1170-yElectronic supplementary material The online version of this article (doi:10.1007/s00253-007-1170-y)contains supplementary material, which is available to authorized users.X.Zhang:J.C.Moore:K.T.Shanmugam:L.O.Ingram(*) Department of Microbiology and Cell Science,University of Florida,Box110700,Gainesville,FL32611,USAe-mail:ingram@K.JantamaDepartment of Chemical Engineering,University of Florida, Gainesville,FL32611,USA(Orlygsson et al.1995)produce alanine from pyruvate and ammonia using an reduced nicotinamide adenine dinucleo-tide (NADH)-linked alanine dehydrogenase (ALD).How-ever,fermentations are slow,and yields from the best natural producers are typically 60%or less because of coproduct formation (Hashimoto and Katsumata 1998;Table 1).Plasmid-borne genes encoding NADH-linked ALD have been tested as an approach to develop improved biocatalysts with varying degrees of success (Table 1).Engineered strains of Zymomonas mobilis CP4expressing the B.sphaericus alaD gene produced low levels of racemic alanine during the anaerobic fermentation of 5%glucose (Uhlenbusch et al.1991).A native chromosomal lactate dehydrogenase gene (ldhA )-deleted strain of Lactococcus lactis containing a mutation in alanine racemase was engineered in a similar fashion and produced 12.6g l −1L -alanine from 1.8%glucose (Hols et al.1999).An Escherichia coli aceF ldhA double mutant containing pTrc99A-alaD plasmid produced 32g l −1racemic alanine in 27h during a two-stage (aerobic and anaerobic)fermentation with a yield of 0.63g alanine g −1glucose (Lee et al.2004).With further gene deletions and process optimization,the racemic alanine titer wasincreasedFig.1Alanine pathway in recombinant E.coli .a Native and recom-binant fermentation pathways.The foreign gene,G.stearothermophilus alaD ,is shown in bold .G.stearothermophilus alaD coding region and transcriptional terminator were integrated into the native ldhA gene under transcriptional control of the ldhA promoter.Solid stars represent deletions of native genes in XZ132.Note that the native biosynthetic route for alanine production is omitted for simplicity.ackA Acetate kinase,adhE alcohol/aldehyde dehydrogenase,alaD alanine dehydro-genase (Geobacillus stearothermophilus XL-65-6),aldA aldehyde dehydrogenase A,aldB aldehyde dehydrogenase B,alr alanine race-mase 1,dadX alanine racemase 2,frd fumarate reductase,gloA glyoxalase I,gloB glyoxalase II,gloC glyoxalase III,ldhA D -lactate dehydrogenase,mdh malate dehydrogenase,mgsA methylglyoxal synthase,pflB pyruvate –formate lyase,ppc phosphoenolpyruvate carboxylase,pta phosphate acetyltransferase.b Coupling of ATP production and growth to NADH oxidation and L -alanine production.Glucose is metabolized to pyruvate,ATP,and NADH.Energy conserved in ATP is utilized for growth and homeostasis,regenerating ADP.NADH is oxidized by alanine formation allowing glycolysis and ATP production to continueT a b l e 1C o m p a r i s o n o f a l a n i n e -p r o d u c i n g s t r a i n sO r g a n i s m s M o d i f i e d p r o p e r t yM e d i a ,s u b s t r a t e a n d p r o c e s s c o n d i t i o n sT i m e (h )A l a n i n e (g l −1)Y i e l d (%)L -A l a n i n ep u r i t y (%)R e f e r e n c e E .c o l i X Z 132I n t e g r a t e d G .s t e a r o t h e r m o p h i l u s a l a D ;Δp f l ,Δa c k A ,Δa d h E ,Δl d h A ,Δm g s A ,Δd a d XM i n e r a l m e d i u m ,b a t c h ,g l u c o s e 120g l −148.0114.095>99T h i s s t u d yA r t h r o b a c t e r o x y d a n s H A P -1M i n e r a l m e d i u m ,t w o -s t a g e f e d -b a t c h ,g l u c o s e 150g l −1120825560.0H a s h i m o t o a n d K a t s u m a t a 1998A .o x y d a n s D A N 75A l a n i n e r a c e m a c e d e f i c i e n tM i n e r a l m e d i u m ,t w o -s t a g e f e d -b a t c h ,g l u c o s e 150g l −1,0.2g l −1D -a l a n i n e120775198H a s h i m o t o a n d K a t s u m a t a 1998E c o l i A L 1(p O B P 1)P l a s m i d w i t h A .o x y d a n s H A P -1a l a D M i n e r a l m e d i u m ,g l u c o s e 20g l −1,l i m i t e d o x y g e n40841N o t r e p o r t e dK a t s u m a t a a n d H a s h i m o t o 1996C o r y n e b a c t e r i u m g l u t a m i c u m A L 107(p O B P 107)P l a s m i d w i t h A .o x y d a n s H A P -1a l a DC o r n s t e e p l i q u o r ,g l u c o s e 200g l −1,4g l −1D L -a l a n i n e ,l i m i t e d o x y g e n 707136>99K a t s u m a t a a n d H a s h i m o t o 1996Z y m o m o n a s m o b i l i s C P 4(p Z Y 73)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D M i n e r a l s a l t s m e d i u m ,s i m p l e b a t c h ,g l u c o s e 50g l −126816N o t r e p o r t e dU h l e n b u s c h e t a l .1991L a c t o c o c c u s l a c t i s N Z 3950(p N Z 2650)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h AR i c h m e d i u m (M 17),g l u c o s e 18g l −117137085–90H o l s e t a l .1999L .l a c t i s P H 3950(p N Z 2650)P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h A ,Δa l rR i c h m e d i u m (M 17),g l u c o s e 18g l −1,0.2g l −1D -a l a n i n e 17N o t k n o w nN o t k n o w n >99H o l s e t a l .1999E .c o l i A L S 887(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δl d h A ,Δa c e FY e a s t e x t r a c t ,t w o -s t a g e b a t c h ,g l u c o s e 50g l −1,a e r o b i c a i r 1l m i n −1273263N o t r e p o r t e d L e e e t a l .2004E .c o l i A L S 929(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δp f l ,Δp p s ,Δp o x B ,Δl d h A ,Δa c e E FY e a s t e x t r a c t a n d c a s a m i n o a c i d s ,t w o -s t a g e b a t c h (a e r o b i c c e l l g r o w t h a n d a n a e r o b i c f e r m e n t a t i o n )223486N o t r e p o r t e d S m i t h e t a l .2006E .c o l i A L S 929(p T r c 99A -a l a D )P l a s m i d w i t h B .s p h a e r i c u s I F O 3525a l a D Δp f l ,Δp p s ,Δp o x B ,Δl d h A ,Δa c e E FY e a s t e x t r a c t a n d c a s a m i n o a c i d s ,t w o -s t a g e f e d -b a t c h (a e r o b i c c e l l g r o w t h a n d a n a e r o b i c f e r m e n t a t i o n )4888100N o t r e p o r t e d S m i t h e t a l .2006to88g l−1in a more complex process with yields ap-proaching the theoretical maximum(Smith et al.2006). However,this strain produced only racemic alanine,utilized multicopy plasmids requiring antibiotic selection,and required complex media with a complex multistage fermen-tation process(Smith et al.2006).In this study,we developed novel biocatalysts that pro-duce chirally pure L-alanine in batch fermentations without using plasmid-containing biocatalysts,antibiotics,or com-plex nutrients.The resulting strains are based on a deriva-tive of E.coli W(strain SZ194)that produces D-lactate (Zhou et al.2006b).The ldhA gene in SZ194was replaced with a single,chromosomally integrated copy of the ALD gene from the thermophile,Geobacillus stearothermophilus XL-65-6(formerly B.stearothermophilus;Lai and Ingram 1993).After additional deletions of alanine racemase (dadX)and methylglyoxal synthase(mgsA)and metabolic evolution,the resulting strain produced L-alanine at high titers(over1M)and yields in batch fermentations using the mineral salts medium.Materials and methodsStrains,plasmids,media,and growth conditionsThe strains and plasmids used in this study are listed in Table2.Strain SZ194was previously engineered from a derivative of E.coli W(ATCC9637)and served as a starting point for constructions(Zhou et al.2006b).G. stearothermophilus XL-65-6(Lai and Ingram1993)was used for cloning the ALD gene.During sequencing of chro-mosomal genes,we discovered a20-year-old error in culture labeling.Strain SZ194,the parent used to construct the alanine strains,is a derivative of E.coli W(ATCC9637). Other constructs for ethanol production and lactate produc-tion that have been reported previously as derivatives of E. coli B are now known to be derivates of E.coli W(ATCC 9637).Primers used in this study are listed in Table3.During strain construction,cultures were grown aerobi-cally at30,37,or39°C in Luria broth(10g l−1Difco tryptone,5g l−1Difco yeast extract,and5g l−1NaCl) containing2%(w/v)glucose or5%(w/v)arabinose. Ampicillin(50mg l−1),tetracycline(12.5mg l−1), kanamycin(50mg l−1),or chloramphenicol(40mg l−1) were added as needed.For initial tests of fermentative alanine production,strains were grown without antibiotics at37°C in NBS mineral salts medium(Causey et al.2004) supplemented with100mM ammonia sulfate,1mM betaine,and2%(w/v)glucose.Fermentation experiments (2–12%sugar)were carried out in NBS medium and AM1 medium(Martinez et al.2007).Broth was maintained at pH 7by the automatic addition of5M NH4OH.Genetic methodsStandard methods were used for genomic deoxyribonucleic acid(DNA)extraction(Qiagen,Valencia,CA),polymerase chain reaction(PCR)amplification(Stratagene,La Jolla CA,and Invitrogen,Carlsbad,CA),transformation,plas-mid extration(Qiagen),and restriction endonuclease diges-tion(New England Biolabs,Ipswich,MA).Methods for foreign gene(alaD)integration and for chromosomal gene (mgsA and dadX)deletion are described below.DNA sequencing was provided by the University of Florida Interdisciplinary Center for Biotechnology Research.The Biocyc and Metacyc databases(Karp et al.2005)were instrumental in the design and completion of these studies. Cloning the alanine dehydrogenase gene alaD from G. stearothermophilus XL-65-6and detection of the enzyme activityThe primers for amplifying alaD from G.stearothermophilus XL-65-6were designed based on the alaD sequence of G. stearothermophilus strain10.The forward primers(5′–3′GGAAAAA GGAGGAAAAAGTG ATGAAGATCGG CATT)included the ribosomal-binding region(bold)and the amino terminus(italicized).The reverse primer(5′–3′GAA GGAGTTGATCATTGTTTAACGAGAGAGG)was down-stream from the putative transcriptional terminator region (Table3).ALD was verified in clones using an activity stain (Kuroda et al.1990).E.coli TOP10F′harboring plasmids containing alaD was grown on Luria–Bertani(LB)plates at 37°C,then transferred to a Whatman7.0-cm filter paper. The filter was immersed in10mM potassium phosphate buffer(pH7.2)and incubated for20min at80°C for lysis of the cells and denaturation of the E.coli proteins.The dried filter paper was assayed in a reaction mixture containing50mM L-alanine,50mM Tris–HCl buffer (pH9.0),0.625mM NAD+,0.064mM phenazine metho-sulfate,and0.24mM nitro blue tetrazolium.The cells with ALD appeared as blue spots on the filter.Integration of alaD into E.coli SZ194The alaD gene was integrated into the chromosomal ldhA gene of SZ194.The fragment(Sma I–Kpn I,1.7kb)con-taining a tet gene flanked by two FRT sites was isolated from pLOI2065and cloned into pLOI4211between a unique Bam HI site(Klenow-treated)and Kpn I site to produce plasmid pLOI4213(6.0kb).In this plasmid,transcription of alaD and tet are oriented in the same direction.The Apa I(treated with T4DNA polymerase to produce a blunt end)–Kpn I fragment(2.2kb)containing alaD and tet was isolated from pLOI4213and cloned into pLOI2395Table2 E.coli strains and plasmids used in this studyRelevant characteristics Source or referenceStrainsSZ194plfB frd adhE ackA deletions Zhou et al.2006bXZ103-110SZ194,ldhA::FRT-tet-FRT::This studyG.stearothermophilus alaDXZ111XZ105,ldhA::G.stearothermophilus alaD This studyXZ112XZ111,metabolic evolution in NBS medium with2%glucose This studyXZ113XZ112,metabolic evolution in NBS medium with5%glucose This studyXZ115XZ113,metabolic evolution in NBS medium with8%glucose This studyXZ121XZ115,mgsA deletion This studyXZ123XZ121,metabolic evolution in NBS medium with8%glucose This studyXZ126XZ123,dadX deletion This studyXZ129XZ126,metabolic evolution in NBS medium with8%glucose This studyXZ130XZ129,metabolic evolution in AM1medium with8%glucose This studyXZ131XZ130,metabolic evolution in AM1medium with10%glucose This studyXZ132XZ131,metabolic evolution in AM1medium with12%glucose This studyPlasmidspCR2.1-TOPO bla kan;TOPO TA cloning vector InvitrogenDatsenko and Wanner2000pKD46Blaγβexo(Red recombinase),temperature conditionalpSC101repliconpFT-A Bla flp,temperature conditional pSC101replicon Posfai et al.1997pEL04cat-sacB targeting cassette Lee et al.2001;Thomason et al.2005 pLOI2224kan;R6K conditional integration vector Martinez-Morales et al.1999pLOI2065bla;FRT-tet-FRT cassette Zhou et al.2003bpLOI2395bla;ldhA franked by two Asc I site Zhou et al.2003apLOI3421 1.8kbp SmaI fragment containing aac Wood et al.2005pLOI4151bla cat;cat-sacB cassette This studyalaD integrationThis studypLOI4211bla kan alaD;alaD(PCR)from G.stearothermophilus XL-65-6cloned into pCR2.1-TOPO vectorpLOI4213bla kan;alaD-FRT-tet-FRT Kpn I-Sma I fragment(FRT-tet-FRT)This studyfrom pLOI2065cloned into Kpn I-BamH I(blunted)site of pLOI4211This studypLOI4214bla kan;ldhA’-alaD-FRT-tet-FRT-ldhA”Apa I(blunted)-Kpn I fragment(alaD-FRT-tet-FRT)from pLOI4213cloned into ldhA at Hinc II-Kpn Isites of pLOI2395This studypLOI4215kan;ldhA’-alaD-FRT-tet-FRT-ldhA”Asc I fragment(ldhA’-alaD-FRT-tet-FRT-‘ldhA)from pLOI4214cloned into Asc I sites of pLOI2224mgsA deletionThis studypLOI4228bla kan;yccT’-mgsA-helD’(PCR)from E.coli W clonedinto PCR2.1-TOPO vectorThis studypLOI4229cat-sacB cassette PCR amplified from pLOI4151(Eco RV digested)cloned into mgsA in pLOI4228This studypLOI4230PCR fragment amplified from pLOI4228(using mgsA-1/mgsA-2primers),kinase treated,and self-ligateddadX deletionThis studypLOI4216bla kan;dadA’-dadX-cvrA’(PCR)from E.coli W clonedinto PCR2.1-TOPO vectorpLOI4218cat-sacB cassette PCR amplified from pLOI4151(Eco RV digested)This studycloned into dadX in pLOI4216This studypLOI4220PCR fragment amplified from pLOI4216(using dadX-4/dadX-5primers),kinase treated,and self-ligated(Hinc II to Kpn I sites)to produce pLOI4214(6.5kb).In this plasmid,ldhA ,alaD ,and tet genes are transcribed in the same direction.The Asc I fragment (4.3kb)containing these three genes was isolated from pLOI4214and cloned into the R6K integration vector pLOI2224to produce pLOI4215(6.2kb).Plasmid pLOI4215contains resistance genes for both tetracycline and kanamycin (Fig.2).The Asc I fragment (4.3kb)containing ldhA ,alaD ,and tet genes was isolated from pLOI4215,further cut by Xmn I to eliminate any remaining uncut plasmid DNA,and electroporated into SZ194containing the Red recombinase plasmid pKD46(Datsenko and Wanner 2000).Integrants were selected for tetracycline resistance,confirmed by sensitivity to kanamycin and ampicillin and by PCR analysis using the primers of ldhA and its neighboring genes ydbH and hslJ (Table 3).Deletion of mgsA and dadX genesA modified method for deleting E.coli chromosomal genes was developed using two steps of homologous recom-bination (Thomason et al.2005).With this method,no antibiotic genes or scar sequences remain on the chromo-some after gene deletion.In the first recombination,part of the target gene was replaced by a DNA cassette containing a chloramphenicol resistance gene (cat )and levansucrase gene (sacB ).In the second recombination,the cat –sacBcassette was removed by selection for resistance to sucrose.Cells containing the sacB gene accumulate levan during incubation with sucrose and are killed.Surviving recombi-nants are highly enriched for loss of the cat –sacB cassette.A new cassette was constructed as a template to facilitate gene deletions.The cat –sacB region was amplified from pEL04(Lee et al.2001;Thomason et al.2005)by PCR using the JM catsacB up Nhe I and JM catsacB down Nhe I primers (Table 3),digested with Nhe I,and ligated into the corresponding site in pLOI3421to produced pLOI4151.The cat –sacB cassette was amplified by PCR using pLOI4151as a template with the cat -up2and sacB -down2primers (Eco RV site included in each primer),digested with Eco RV ,and used in subsequent ligations.The mgsA gene and neighboring 500-bp regions (yccT ′–mgsA –helD ′,1,435bp)were amplified using the mgsA -up and mgsA -down primers and cloned into the pCR 2.1-TOPO vector (Invitrogen)to produce plasmid pLOI4228.A 1,000-fold diluted plasmid preparation of this plasmid served as a template for inside-out amplification using the mgsA -1and mgsA -2primers (both within the mgsA gene and facing outward).The resulting 4,958-bp fragment containing the replicon was ligated to the Eco RV-digested cat –sacB cassette from pLOI4151to produce pLOI4229(Fig.3a).This 4,958-bp fragment was also used to construct a second plasmid,pLOI4230(Fig.3b),by phosphorylation and self-ligation.In pLOI4230,the central region of mgsA is deleted (yccT ′–mgsA ′–mgsA ″–helD ′).After digestion of pLOI4229and pLOI4230with Xmn I (within the vector),each served as a template for amplifica-tion using the mgsA -up and mgsA -down primers to produce linear DNA for integration step 1(yccT ′–mgsA ′–cat –sacB –mgsA ″–helD ′)and step II (yccT ′–mgsA ′–mgsA ″–helD ′),respectively.After electroporation of the step 1fragment into XZ115containing pKD46(Red recombinase)and 2h ofTable 3Primers used in this study Primers SequencealaD -forward GGAAAAAGGAGGAAAAAGTGATGAA GATCGGCATTalaD -reverse GAAGGAGTTGATCATTGTTTAACGA GAGAGGldhA -forward AGTACCTGCAACAGGTGAAC ldhA -reverse CAGGCGACGGAATACGTCAT ldhA -up (ydbH )CTGATAACGCAGTTGCTGGA ldhA -down (hslJ )TTCATTAAATCCGCCAGCTTJM catsacB up NheI TTAGCTAGCATGTGACGGAAGATC ACTTCGJM catsacB down NheI CCGCTAGCATCAAAGGGAAAACTGT CCATATcat -up2AGAGAGGATATCTGTGACGGAAGAT CACTTCGsacB -down2AGAGAGGATATCGAATTGATCCGGT GGATGACmgsA -up CAGCTCATCAACCAGGTCAA mgsA -down AAAAGCCGTCACGTTATTGG mgsA -1AGCGTTATCTCGCGGACCGT mgsA -2AAGTGCGAGTCGTCAGTTCC dadX -up AGGCTACTCGCTGACCATTC dadX -down GGTTGTCGGTGACCAGGTAG dadX -4TGGGCTATGAGTTGATGTGC dadX -5CTGTATCGGACGGGTCATCTFig.2Integration vector used for chromosomal insertion of G.stearothermophilus alaD into E.coli ldhA .Sequence encoding the N-terminal and C-terminal regions are designated ldhA ′and ldhA ″,respectivelyincubation at 30°C to allow expression and segregation,recombinants were selected for chloramphenicol (40mg l −1)and ampicillin (50mg l −1)resistance in Luria broth at 30°C (18h).Three clones were selected,grown in Luria broth containing ampicillin and 5%(w/v)arabinose (to induce expression of red recombinase),and prepared for electro-poration.After electroporation with the step 2fragment,cells were incubated at 30°C for 4h and then transferred into a 250-ml flask containing 100ml of modified LB (100mM 3-(N -morpholino)propanesulfonic acid [MOPS]buffer added and NaCl omitted)containing 10%sucrose.After overnight incubation (30°C),clones were selected on modified LB plates (no NaCl;100mM MOPS added)containing 6%sucrose (39°C,16h).Resulting clones were tested for loss of ampicillin and chloramphenicol resistance.Construction was confirmed by PCR using the mgsA-up/down primer set.A clone containing a deletion in the central region of mgsA was selected and designated XZ121.The dadX gene was deleted in a manner analogous to that used to delete the mgsA gene.Primers for dadX deletion are shown in Table 3,and the corresponding plasmids are shown in Table 2.FermentationNBS mineral salts medium (Causey et al.2004)with 1mM betaine (Zhou et al.2006a )was used in the initial fermentation (pH 7.0).Preinoculum was grown by inocu-lating three colonies into a 250ml flask (100ml NBS medium,2%glucose,and 100mM ammonium sulfate).After 16h (37°C,120rpm),this preinoculum was diluted into 500-ml fermentation fleakers containing 300ml NBS medium (2–8%glucose,100mM ammonium sulfate,and 1mM betaine)with 33mg cell dry weight (CDW)l −1.In early experiments,pH was maintained at 7.0by automat-ically adding 2M potassium hydroxide.In later experi-ments,5M ammonium hydroxide was used to maintain pH,and a low salt medium,AM1(Martinez et al.2007),was used to replace the NBS medium for fermentation (8–12%glucose).AM1medium contains much less salt and has been optimized for E.coli .Metabolic evolutionCells from pH-controlled fermentations were serially transferred at 24-h intervals to facilitate metabolic evolution through competitive,growth-based selection (Fig.1b).At the beginning,sequentially transferred cultures were inoc-ulated with an initial density of 33mg CDW l −1.As growth increased,the inoculum was changed to a 1:100dilution and subsequently to a 1:300dilution.Periodically,clones were isolated from these experiments,assigned new strain designations,and frozen for storage.AnalysesCell mass was estimated by measuring the optical density at anic acids and glucose concentrations were mea-sured by high-performance liquid chromatography (HPLC,Underwood et al.2002).Analysis of fermentation products by mass spectroscopy and amino acid analyzer were provided by the University of Florida Interdisciplinary Center for Bio-technology Research.Alanine was found to be the predominant product.The alanine concentration and isomeric purity were further measured by HPLC using the Chiralpak MA(+)chiral column (Chiral Technologies,West Chester,PA).ResultCloning of the alanine dehydrogenase geneALD is found in Bacillus (and Geobacillus )species where it plays a pivotal role in energy generation during sporulation (Ohashima and Soda 1979;Kuroda et al.1990).ALD from B.sphaericus IFO3525has beenwidelyFig.3Plasmids used to delete mgsA .Plasmid pLOI4229(a )was used to delete the mgsA gene and insert the cat-sacB cassette in the first recombina-tion step.Plasmid pLOI4230(b )was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence.Se-quence encoding the N-terminal and C-terminal regions are des-ignated mgsA ′and mgsA ″,respectivelyused with varying degrees of success to engineer alanine production in recombinant bacteria(Uhlenbusch et al. 1991;Hols et al.1999;Lee et al.2004;Smith et al.2006). Selection of the B.sphaericus IFO3525is presumed to be due in part to the high specific activity(Ohashima and Soda 1979).In contrast,we have selected a thermostable ALD from the thermophile,G.stearothermophilus XL-65-6, based on our prior experience in expressing genes from this organism in recombinant E.coli(Burchhardt and Ingram 1992;Lai and Ingram1993;Lai and Ingram1995).The ribosomal-binding region,coding region,and tran-scriptional terminator of alaD were amplified from G. stearothermophilus XL-65-6and sequenced(EF154460in GenBank).The deduced amino acid sequence was identical to that reported for Geobacillus kaustophilus HTA426and very similar to G.stearothermophilus strain10(99%iden-tity)and G.stearothermophilus strain IFO12550(94% identity).The nucleotide sequence(65%identity)and the deduced ALD amino acid sequence(74%identity)were quite different from the B.sphaericus IFO3525gene,the gene pre-viously used for alanine production in recombinant bacteria.Modification of E.coli W for homoalanine productionE.coli W strain SZ194(pflB frdBC adhE ackA)was previously constructed to produce only D-lactic acid.All major fermentation pathways except lactate have been blocked in this strain by gene deletions(Fig.1a).To convert this strain to the production of alanine,part of the native ldhA-coding region was replaced by a DNA fragment containing the ribosomal-binding region,coding region,and transcriptional terminator of alaD from G. stearothermophilus XL-65-6.The promoterless alaD was oriented in the same direction as ldhA to allow expression from the native ldhA promoter(Fig.2).After electroporation,approximately500colonies were recovered with tetracycline resistance and sensitivity to kana-mycin,consistent with a double-crossover event.These colo-nies were further examined by PCR using ldhA forward and reverse primer set(Table3).Only eight colonies of the500 tested were correct based on an analysis of PCR fragments. These eight colonies were further verified using primer sets for alaD,ldhA forward and alaD reverse,alaD forward and ldhA reverse,and ldhA outside primers(Table3)and de-signated XZ103,XZ104,XZ105,XZ106,XZ107,XZ108, XZ109,and XZ110,respectively.These eight strains were initially tested in15-ml screw-cap tubes containing NBS medium with2%glucose and100mM ammonium sulfate, which were filled to the brim.Strain XZ105appeared to grow faster than the other strains(37°C for48h)and was selected for further development.XZ105was transformed with pFT-A,which contains an inducible flippase(FLP)recombinase(Martinez-Morales et al.1999;Posfai et al.1997).The chromosomal FRT-flanked tet gene in XZ105was removed by inducing the FLP recombinase.After growing in39°C to eliminate the temperature-sensitive plasmid pFT-A,resulting strain was designated XZ111.Expression of G.stearothermophilus alaD in XZ111is transcriptionally regulated by the ldhA promoter,the same promoter that regulates the production of lactate dehydrogenase(dominant fermentation pathway) in native E.coli.pH-controlled batch fermentation for alanine production Alanine production by strain XZ111was tested in500-ml fermentation vessels containing300ml NBS medium, 20g l−1glucose,100mM ammonium sulfate,and1mM betaine.Broth pH was automatically controlled by adding 2N potassium hydroxide.After96h,181mM alanine was produced.The alanine yield from total glucose was 81%(g/g),and84%based on glucose that had been metabo-lized.The chiral purity of L-alanine was96.1%(Table4). Very low levels of other products(lactate,succinate,ace-tate,ethanol)were present,typically below1mM.This result demonstrated that the integrated G.stearothermophilus alaD gene as a single chromosomal copy under the control of the native ldhA promoter can provide sufficient levels of ALD to support E.coli growth from the production of alanine as the sole fermentation product.Metabolic evolution of strain XZ111Although XZ111could accumulate alanine as the primary product,incubation times were long,and volumetric productivity was limited.When using a high-glucose concentration(80g l−1),growth and alanine productivity were further reduced(Table4).In this strain,adenosine triphosphate(ATP)production and growth are tightly coupled to NADH oxidation and alanine production by ALD(Fig.1b).This coupling provided a basis for strain improvement by selecting for increased growth during serial cultivation,i.e.,metabolic evolution.Cells with increased growth because of spontaneous mutations will successively displace their parents while coselecting for increased alanine productivity.Serial transfers of XZ111were carried out at24-h intervals in NBS mineral salts medium with1mM betaine.Cultures were first transferred in the medium containing20g l−1 glucose,and the pH was controlled by automatically adding 2N potassium hydroxide.However,after ten transfers to strain XZ112,little improvement was observed(data not shown).Because ammonia is essential for alanine pro-duction,it was thought that ammonia may be limiting for fermentation.Two normals potassium hydroxide containing 1N ammonia carbonate and5N ammonia hydroxide alone。
upadacitinib质量标准
对于upadacitinib的质量标准，我们需要从多个方面进行考量和描述。
首先，我们将从药物的化学性质、理化性质、纯度、稳定性等方面展开详细的描述，以确保upadacitinib的质量标准在生产和使用过程中得到充分的满足。
1. 化学性质upadacitinib是一种小分子化合物，其化学名称为(3R,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin -8-yl)pyrrolidin-2-one。
该化合物的结构式如下：[图片]对于upadacitinib的化学性质，我们需要确定其分子量、分子式、化学结构等信息，并且需要特别关注其可能存在的同系物或杂质的种类和含量。
此外，还需要对其在不同溶剂中的溶解性进行评估，以确保其在制剂过程中的适用性和稳定性。
2. 理化性质upadacitinib的理化性质是评价其质量的重要指标之一。
这包括了其外观形态、熔点、沸点、溶解度、pH值等各项指标。
这些数据可以帮助我们了解upadacitinib在不同条件下的物理状态和化学性质，为其生产工艺和质量控制提供重要参考。
3. 纯度upadacitinib的纯度是其质量标准中至关重要的一环。
通过高效液相色谱（HPLC）等技术手段，我们需要对upadacitinib 进行杂质和同系物的检测和定量，确保其符合国际药典或公司内部标准的要求。
另外，残留溶剂的含量也需要进行严格的监控和限制。
4. 稳定性upadacitinib的稳定性是其在生产、运输和使用过程中必须考虑的重要问题。
这包括了其在不同温度、湿度、光照等条件下的稳定性表现，以及在制剂中的保存稳定性。
通过进行加速稳定性研究和长期稳定性研究，我们可以获得upadacitinib在不同条件下的降解规律和稳定性表现，为其合理的保存和使用提供依据。
5. 其他此外，针对upadacitinib的质量标准，还需要考虑其微生物限度、重金属含量、残留农药等方面的要求，以确保其在使用过程中对人体的安全性。
阿拉丁癸二酸二丁酯产品安全技术说明书
GHS07:感叹号3成分/组成信息 3.1物 质分子式:C18H34O4； [-(CH2)4CO2(CH2)3CH3]2分子量 :314.46 g/mol成分 (单一物质)浓度癸二酸二丁酯Dibutyl sebacateCAS No. 109-43-3EC-编号203-672-598%4急救措施4.1必要的急救措施描述一般的建议请教医生。
出示此安全技术说明书给到现场的医生看。
如果吸入如果吸入,请将患者移到新鲜空气处。
如果停止了呼吸,给于人工呼吸。
请教医生。
在皮肤接触的情况下用肥皂和大量的水冲洗。
请教医生。
在眼睛接触的情况下用大量水彻底冲洗至少15分钟并请教医生。
如果误服切勿给失去知觉者从嘴里喂食任何东西。
用水漱口。
请教医生。
4.2最重要的症状和影响，急性的和滞后的据我们所知，此化学，物理和毒性性质尚未经完整的研究。
4.3及时的医疗处理和所需的特殊处理的说明和指示无数据资料5消防措施5.1灭火介质火灾特征无数据资料灭火方法及灭火剂用水雾,耐醇泡沫,干粉或二氧化碳灭火。
5.2源于此物质或混合物的特别的危害碳氧化物5.3救火人员的预防如必要的话,戴自给式呼吸器去救火。
5.4进一步的信息无数据资料6泄露应急处理6.1人员的预防,防护设备和紧急处理程序使用个人防护设备。
防止吸入蒸汽、气雾或气体。
保证充分的通风。
将人员撤离到安全区域。
6.2环境预防措施不要让产物进入下水道。
6.3抑制和清除溢出物的方法和材料用惰性吸附材料吸收并当作危险废品处理。
存放进适当的闭口容器中待处理。
6.4参考其他部分丢弃处理请参阅第13节。
7安全操作与储存7.1安全操作的注意事项避免接触皮肤和眼睛。
防止吸入蒸汽和烟雾。
一般性的防火保护措施。
7.2安全储存的条件,包括任何不兼容性贮存在阴凉处。
容器保持紧闭，储存在干燥通风处。
7.3特定用途无数据资料8接触控制/个体防护8.1控制参数最高容许浓度成分 CAS No. 值控制参数基准癸二酸二丁酯Dibutyl sebacate 109-43-3PC-TWA无数据资料 《工作场所有害因素职业接触限值》国家标准中的工作场所时间加权平均容许浓度无数据资料无数据资料 无数据资料8.2暴露控制适当的技术控制按照良好工业和安全规范操作。
食品化学
食品化学dorystoechas脚叶提取物中抗氧化活性成分和脯氨酸的含量文章信息文章历史：2007年8月7日收到2008年3月18日收到修改2008年3月31日接收关键词：dorystoechas脚chalba茶抗氧化活性DPPH脯氨酸摘要dorystoechas脚（D脚）是土耳其安塔利亚省特有的单子叶植物。
D .脚叶子在当地是用来做茶的,被称为“chalba茶”。
乙醚（E），乙醇（A），和水（W）是用于连续制备干燥D脚叶提取物的溶剂。
热水提取物（S）是指直接用沸水提取植物的成分。
提取物的抗氧化活性成分用硫氰酸铁（FTC）和DPPH清除自由基的方法检测。
乙醚提取物用FTC的方法测定表现出最大的抗氧化活性，而热水提取物中DPPH清除自由基活性最低的IC 50值为（6.17±0.531g /ml）。
用Folin –Ciocalteu方法和热水提取法提取，总酚中大约含有提取物中有效酚类的最大量为（554.17±20.83mgGAE/g）。
乙醇提取物中有效成分含量和DPPH清除自由基活性在最大值和IC50之间有逆性相关（R 2 =0.926）的关系。
降低提取物的耗能，增加提取物的浓度在一定程度上可以减小依赖性。
在20到25mg/ml浓度之间，热水提取物比同等量的抗坏血酸有较高的还原能力。
在减低耗能与浓度的线性关系中发现，乙醇、水和乙醚提取物（R2>0.95）依次含有相对高水平的脯氨酸。
结果表明，D.脚可以提供一个自然来源的抗氧化剂和脯氨酸。
1、序言植物可以产生范围广泛的化学物质，包括“代谢产物”，当被人吃掉可能有利于健康。
在动物中许多植物的次生代谢产物可以作为抗氧化剂，另一方面，这些植物源的代谢产物可通过抑制脂质氧化来防止食品变质。
抗氧化作用是几个不同的化学事件的组合，如金属螯合；淬火自由基中氢提供的自由基的酚醛树脂群体；氧化的非传播自由基；氧化还原电位；酶抑制剂（威廉姆森，罗德，公司，1999）。
加速溶剂萃取-超高效液相色谱-串联质谱法测定大枣中3种五环三萜酸
食品与药品Food and Drug2021年第23卷第1期17加速溶剂萃取-超高效液相色谱-串联质谱法测定大枣中3种五环三祜酸张萍，何婷，王颖，胡克特，顾丁，陈荣祥**(遵义医科大学基础医学院，贵州遵义563000)摘要：目的建立加速溶剂萃取-超高效液相色谱-串联质谱法(ASE-UPLC-MS/MS)同时测定大枣中桦木酸、齐墩果酸和熊果酸的方法。
方法样品釆用ASE提取，优化提取条件，并与超声辅助提取法进行比较。
优化 后的提取条件为：以80%甲醇为提取溶剂，提取温度100-C,静态萃取时间15min,萃取1次。
提取液釆用Waters ACQUITY BEH C18色谱柱分离，以乙ffi-15mmol/L乙酸钱(pH9.3)为流动相，梯度洗脱，经UPLC-MSZMS仪，釆用电喷雾电离源，负离子模式下多反应监测模式检测。
结果桦木酸、齐墩果酸、熊果酸在0.5~10 mg/L范围内，浓度与峰面积线性关系良好，相关系数大于0.9900。
不同浓度3种化合物的加标回收率93.6%〜101.7%,相对标准偏差在1.18%~6.83%之间。
结论此法简单快速、准确稳定、重复性好，可用于大枣中桦木酸、齐墩果酸、熊果酸的含量测定。
关键词：加速溶剂萃取；超高效液相色谱；串联质谱法；大枣中图分类号：R284.1文献标识码：A文章编号：1672-979X(2021)01-0017-06DOI：10.3969/j.issn.l672-979X.2021.01.004Simultaneous Determination of Three Pentacyclic Triterpenic Acids in Jujubae Fructus byAccelerated Solvent Extraction-UPLC-MS/MSZHANG Ping,HE Ting,WANG Ying,HU Ke-te,GU Ding,CHEN Rong-xiang(School of B asic Medical Sciences,Zunyi Medical University,Zu^yi563000,China)Abstract:Objective To establish a method for the simultaneous determination of betulinic acid,oleanolic acid and ursolic acid in Jujubae fructus by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)with accelerated solvent extraction(ASE).Methods The extract parameters of A SE were optimized and the efficiency was compared with the ultrasound-assisted extraction method.The optimum extraction conditions were as follows:80%methanol was selected as extraction solvent,oven temperature was100°C,the static extraction time was 15min and one extraction cycle was adopted.A waters ACQUITY BEH C18(2.1mmx]00mn,1.7“m)column was used as the stationary phase,acetonitrile and ammonium acetate solution(15mmol/L,pH9.3)was used as the mobile phase.Mass detection was conducted by electrospray ionization in negative ion multiple reaction monitoring mode. Results The calibration curves were linear over a concentration range of0.5-10mg/L for betulinic acid,oleanolic acid and ursolic acid.The correlation coefficients were greater than0.9900.The recoveries of different spiked levels were between93.6%and101.7%,with RSDs between1.18%and6.83%.Conclusion The method is simple,rapid,收稿日期：2020-09-04基金项目：国家自然科学基金(31660131,81760652)；贵州省联合基金(黔科合J字LKZ[2013]17号)；遵义医学院博士启动基金(F-568)作者简介：张萍，硕士研究生，研究方向：药用植物开发与利用E-mail:******************通讯作者：陈荣祥，教授，博士，研究方向：药用植物开发与利用E-mail:*************************18食品与药品Food and Drug2021年第23卷第1期accurate,stable and reproducible.It can be used for the determination of betulinic acid,oleanolic acid and ursolic acid in Jujubae f ructus.Key Words:accelerated solvent extraction;ultra-high performance liquid chromatography;tandem mass spectrometry; Jujubae f ructus.大枣为鼠李科植物枣树（Ziziphus jujuba Mill.）的干燥成熟果实，不仅作为食物，还是传统中医药中的常用药材。
沙奎那韦
谢谢观看
联合治疗可以使AIDS合并症或垂危状态的危险性减少53%，死亡率减少72%。这与治疗18个月后AIDS合并症或 死亡率由29.4%降至16.0%是相符的；同样，单纯死亡率由8.6%降至4.1%。在3个治疗组中，平均疗程为11~13个 月，平均随访时间是17个月。
该研究中，所有治疗组CD4细胞基线计数平均为156~176/立方毫米。16周后（DAVG16），沙奎那韦联合ddc 治疗组CD4细胞增加26/立方毫米，血浆病毒载量减少0.6log10RNA拷贝/毫升。16周时，CD4细胞平均值增加47/ 立方毫米。12周时，血浆病毒载量平均值降低0.7log10RNA拷贝/毫升。
与核苷类似物（齐多夫定等）不同，沙奎那韦直接作用于病毒靶酶，不需经代谢激活，对静止细胞也有潜在 作用。在10-10摩尔/升浓度下，沙奎那韦对淋巴母细胞株和单核细胞株以及被实验室病毒株或临床分离的HIV-1 感染的淋巴细胞和单核细胞的起始培养有作用。
实验室细胞培养结果显示，沙奎那韦在与其他逆转录酶抑制剂（包括AZT（齐多夫定）、ddc（扎西他滨）、 ddI（去羟肌苷）进行两联或三联治疗HIV-1感染时，有附加的协同抗病毒作用，但毒性并不增加 。
机体对静注沙奎那韦6、36、72毫克后清除率很高，为1.14升/小时/千克（CV12%），略高于肝血流，并为常 数。体内存留时间平均为7小时。
适应症
沙奎那韦可与其他抗逆转录病毒药物联合使用治疗成人HIV-1感染 。
用法与用量
1
标准剂量
2
剂量调整
3
不良反应
4
禁忌
5
药物相互作用
成人及16岁以上儿童：推荐方案是与核苷类似物联合用药，餐后2小时内服用沙奎那韦600毫克，每天3次。 联合使用的抗逆转录病毒药物的剂量参考处方手册。与其他蛋白酶抑制剂合用时，沙奎那韦应减量（见【药物相 互作用】）。与其他蛋白酶抑制剂一样，强烈推荐按医嘱服药。
氢溴酸达非那新
适应证
氢溴酸达非那新用于膀胱过度刺激引起的尿频、尿急、尿失禁。
禁忌证
1.对氢溴酸达非那新及其中成分过敏者禁用。 2.尿潴留、胃潴留及未控制的闭角型青光眼患者禁用。 3.重度肝功能损害患者不推荐使用。
注意事项
1.由于尿潴留的可能，有明显膀胱尿道阻塞症状的患者使用时应谨慎。 2.氢溴酸达非那新具有抗胆碱作用，能降低胃肠道动力，胃肠道阻塞性疾病患者有胃潴留的可能，使用时应 谨慎。严重便秘、溃疡性结肠炎和重症肌无力患者慎用。 3.已控制的闭角型青光眼患者慎用。 4.氢溴酸达非那新生殖毒性分级为C，只有当对母体的益处高于对胎儿的危险时方可用于孕妇。 5.氢溴酸达非那新可经大鼠乳汁分泌，尚不知氢溴酸达非那新是否经人乳汁分泌，哺乳期妇女应慎用。
用法用量
口服，推荐剂量为7.5mg，1次/d，整片服下，不得嚼碎、掰开或压碎，可单服或与食物同服。根据个人临床 反应，剂量可增至15mg。中度肝功能损伤患者及与CYP3A4抑制剂（如酮康唑、伊曲康唑、利托那韦、奈非那韦、 克拉霉素、奈法唑酮）同服时，剂量不得超过7.5mg。
药物相互作用
1.氢溴酸达非那新主要经CYP2D6和CYP3A4代谢，CYP3A4抑制剂（酮康唑、伊曲康唑、利托那韦、奈非那韦、 克拉霉素、奈法唑酮）可使氢溴酸达非那新代谢减少，日剂量不应超过7.5mg。
尿失禁治疗药物是一个潜力巨大但尚未完全开发的市场，临床特征均是在24h内需要小便数不少于十次。据 世界卫生组织(WHO)有关人员估计，全球约有10%～15%中年人(50岁以下)和40%～70%老年人不同程度地受到此病 困扰。膀胱过动症一般没有神经源性损伤或疾病，可由膀胱的快速充盈、体位改变、甚至行走、咳嗽诱发。估计 全世界约有4～5亿名尿失禁患者，女性的发生率为男性的2倍。男性的发生率随着年龄的增长而升高，是一种常 见和令人痛苦的疾病。（另有一组数据估计世界7个主要国家受影响的人群达1.54亿，其中0.73亿人被分类为明 显尿失禁症。）。
高效液相色谱-串联质谱法检测泮托拉唑钠原料药中的水合肼
·药物研发·高效液相色谱-串联质谱法检测泮托拉唑钠原料药中的水合肼赵会明 张振洋 樊华军［英格尔检测技术服务（上海）有限公司 上海 201100］摘要建立了泮托拉唑钠原料药中的基因毒性杂质水合肼的高效液相色谱-串联质谱（LC-MSMS）检测方法。
采用反相色谱，以水-乙腈（含0.1%甲酸）为流动相，梯度洗脱，流速0.5 mL/min，以ESI正离子多反应监测（MRM）模式进行质谱检测。
结果显示，水合肼的检测限和定量限可达到0.23、0.47 ng/mL，其在0.47~9.37 ng/mL浓度范围内线性关系良好（r=0.999 9），准确度试验中低、中、高浓度回收率均在81.6%~90.9%之间。
在3批次泮托拉唑钠原料药中均未检出水合肼。
关键词高效液相色谱-串联质谱法基因毒性杂质泮托拉唑钠水合肼痕量检测中图分类号：R917; O657 文献标志码：A 文章编号：1006-1533(2022)11-0072-04引用本文 赵会明, 张振洋, 樊华军. 高效液相色谱-串联质谱法检测泮托拉唑钠原料药中的水合肼[J]. 上海医药, 2022, 43(11): 72-75.Determination of hydrazine hydrate in pantoprazole sodium by high performance liquid chromatography-tandem mass spectrometryZHAO Huiming, ZHANG Zhenyang, FAN Huajun[ICAS Testing Technology Service (Shanghai) CO., LTD., Shanghai 201100, China]ABSTRACT To establish a high-performance liquid chromatography-tandem mass spectrometry (LC-MSMS) method for the determination of hydrazine hydrate in active pharmaceutical ingredient (API) pantoprazole sodium. HPLC was carried out by reverse chromatography using water-acetonitrile containing 0.1% formic acid as flow phase and gradient elution at a flow rate of 0.5 mL/min. Mass spectrometry was performed with multi-reaction monitoring (MRM) in positive ESI mode. The detection and quantitative limits of hydrazine hydrate reached 0.23, 0.47 ng/mL and hydrazine hydrate showed good linear relationship in the range of 0.47-9.37 ng/mL (r=0.999 9). The recoveries of samples at low, medium and high-level concentrations reached81.6% to 90.9% in the accuracy experiment. No hydrazine hydrate was detected in 3 batches of pantoprazole sodium.KEY WORDS HPLC-tandem mass spectrometry; genotoxic impurities; pantoprazole sodium; hydrazine hydrate; trace determination上消化道出血是近年的临床疾病中常见且多发的一种疾病，其临床表现为呕血、黑便等，如得不到及时有效治疗，可能引发失血性休克。
盐酸特比萘芬
非常罕见：已报告的有血液系统疾病如中性粒细胞减少症，粒细胞缺乏症或血小板减少症
非常罕见，已报告的有脱发，尽管病因成分过敏者禁用
【用法用量】根据感染的严重程度和适应症调整疗程。
口服，成人每次0．25克，每日一次，疗程如下：皮肤感染的疗程：手足癣[趾(指)间型和跖型]：2—6周；体癣、股癣：2—4周：皮肤患珠菌病：2—4周。在真菌学治愈几周，才可见到皮肤外观完全正常以及感染症状消失。
成人：每次1片（0.25g）每天一次
3．相互作用研究证明，特比萘芬几乎不影响经由细胞色素P450酶系代谢的药物(如环孢素、D860或口服避孕药)的清除。然而，使用口服避孕药的妇女应慎用本品，因为极少数人可能月经失调。此外，肝药酶诱导药(如利福平等)可加速特比萘芬的血浆清除，肝药酶抑制药(如西米替丁等)则可抑制其清除，故如果需要用以上药物，则需将特比萘芬的剂量作适当调整。
青少年，体重＞40kg（通常年龄＞12岁）：每次1片（0.25g）每天一次
儿童，体重20-40kg（通常年龄5-12岁）：每次半片（0.125g）每天一次
儿童，体重＜20kg 通常年龄＜5岁：关于此组病人，从对照试验中获得的资料非常有限，所以药物只有在没有其它可选择的治疗方法以及潜在的治疗疗效益大于可能的危险情况才可使用。
注意事项：
1．肝或肾功能不全(肌酐清除率<50ml／分，血清肌酐>300u mol／升)者，特比萘芬剂量应减少50％(见“不良反应”)。
2．妊娠与哺乳动物研究显示，本品对胎儿和生育力无不良影响。由于本品用于孕妇的经验极有限，因此，应权衡利弊，原则上孕妇不应使用。特比萘芬可经乳汁排泄，故接受特比萘芬口服治疗的母亲不应哺乳。
欧洲药典7.5版
INDEX
To aid users the index includes a reference to the supplement in which the latest version of a text can be found. For example : Amikacin sulfate...............................................7.5-4579 means the monograph Amikacin sulfate can be found on page 4579 of Supplement 7.5. Note that where no reference to a supplement is made, the text can be found in the principal volume.
English index ........................................................................ 4707
Latin index ................................................................................. 4739
EUROPEAN PHARMACOPபைடு நூலகம்EIA 7.5
Index
Numerics 1. General notices ................................................................... 7.5-4453 2.1.1. Droppers...................
【药品名】氢醌【英文名】Hydroquinone
【药品名】氢醌
【英文名】Hydroquinone
【别名】对苯二酚；鸡纳酚；斑必治
【剂型】软膏、乳膏：2%～5%，15g/支。
【药理作用】本品由苯胺氧化成对苯醌，再经还原制成，为脱色剂。
它能阻断酪氨酸酶催化酪氨酸转变成二羟基苯丙氨酸(DPA)，从而抑制了黑色素的生物合成，但不破坏黑素细胞及已形成的黑色素。
【药动学】
【适应症】用于黑变病、黄褐斑、雀斑及色素沉着等。
【禁忌症】
【注意事项】本品极易氧化变色，可适当加入抗氧化剂(如0.1%亚硝酸钠等)，应避光保存。
只供外用。
【不良反应】对皮肤、黏膜有一定刺激性，浓度适宜，不良反应少。
【用法用量】2%～5%软膏或乳膏外用涂患处，2次/d。
【药物相互作用】
【专家点评】。
盐酸阿考替胺
盐酸阿考替胺及制剂一、化合物大体信息英文名AcotiamideHydrochloride HydrateAcotiamidehydrochlorideAcotiamide产品名称阿考替胺盐酸盐三水合物阿考替胺盐酸盐阿考替胺分子结构分子式分子量CAS 登录号773092-05-0二、产品信息【药品名称】盐酸阿考替胺【英文名称】acotiamide hydrochloride hydrate【商品名】Acofide【中文化学名】：N-[2-(二异丙基氨基)乙基]-2-[(2-羟基-4,5-二甲氧基苯甲酰)氨基]-1,3-噻唑-4-羧酸胺盐酸盐三水合物【英文化学名】：N-{2-[Bis(1-methylethyl)amino]ethyl}-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]thiazole-4-carboxamidemonohydrochloride trihydrate【化学结构】：【分子式】：【分子量】：【CAS】：773092-05-0【性状】白色或类白色结晶性粉末【熔点】154℃-156℃【溶解度】本品在DMF中溶解，甲醇中略溶，乙醇和水中微溶，丙酮中极微溶，乙酸乙酯中不溶。
【pka】[药理作用及特点] 阿考替胺最先由日本Zeria新药工业株式会社研发，后由Astellas 制药与Zeria药业在日本一路推出。
阿考替胺口服片剂在消化道主如果通过抑制乙酰胆碱酯的机制起作用，可增进胃动力、改善胃容纳障碍、增强胃底扩张。
最新数据显示，西方国家普通人群消化不良症状已接近40%，并显著降低了生活质量。
其中小部份人群由胃溃疡引发，可通过根治幽门螺杆菌进行医治，约20%的有症状人群属于胃食道逆流疾病，可通过质子泵抑制剂有效医治，但大多数消化不良人群属于FD，对于FD的医治目前仍具有挑战性。
【注册分类】化药3+3类【剂型规格】100mg【制剂】片剂【适应症】用于医治功能性消化不良（FD）【用法用量】一天三次，一次100mg，饭前服用。