Determination of Squalene by Ultra Performance Liquid Chromatography

应用超高效液相色谱法,建立了角鲨烯的测定方法。采用uPlC bEH C18(2.1MMx50MM,1.7μM)色谱柱考察了流动相有机相比例、流速及柱温对角鲨烯样品分离的影响,确定了最佳色谱条件:等度洗脱,流动相为甲醇-水(95∶5,体积比),流速0.4M l/MIn,柱温35℃,检测波长210nM。该方法下,角鲨烯在0.2-10μg/M l范围内浓度和面积呈现良好的线性关系,相关系数r为0.9998;检测限为0.1ng,定量限分别为0.25ng,精密度试验rSd(n=6)为0.36%,平均回收率为100.82%(rSd=0.99%)。本方法快速、简单、可靠、灵敏、重复性好,可用于角鲨烯有关样品的快速检测。The article is researching determination of squalene by Ultra performance liquid chromatography(UPLC).The analysis was performed on an Acquity UPLC BEH C18 column(2.1mm×50mm,1.7μm).The influence of flow rate,the proportion of organic phase in mobile phase,and column temperature on the separation of squalene was comprehensively studied.The optimal separation condition was as follows:a mobile phase consisting of methanol and water(95∶5,v/v) with a isocratic elution profile,UVdetection wavelength at 210 nm,0.4 m L/min of flow rate for mobile phase,35℃ of column temperature.On the above condition,good linear was observed in the linear range of 0.2-10 μg/m L,and the correlation coefficient r was 0.9998.The limits of detection(S/N=3) were 0.1ng and the limits of quantification(S/N=10) were 0.25 ng.The average recovery rates was 100.82%(RSD=0.99%).The RSD of repetition was 0.36%(n=6).This method was simple,accurate and sensitive with a good reproducibility.It is suitable for fast detection of squalene.厦门海洋研究开发院共建项目(2014); 海洋生物技术产业化中试技术研发公共服务平台(12PZP001SF10); 广东海洋经济发展区域示范项目(GD2012-D01-001

Determination of Astaxanthin by High Performance Liquid Chromatography with Photodiode Array Detector

建立虾青素含量测定的高效液相色谱–光电二极管阵列法。采用PurOSPHEr STAr rP 18(4.6 MMx250MM,5μM)色谱柱,以甲醇–水(体积比为95∶5)为流动相,流速1.0 Ml/MIn,检测波长为482 nM,柱温为30℃,进样量为20μl。在所选定的液相色谱条件下,虾青素主峰与其它杂质峰分离良好,虾青素在0.2~16μg/Ml范围内线性良好,线性相关系数r=0.999 9,检出限为0.01μg/Ml,测定结果的相对标准偏差为0.42%(n=6),平均回收率为100.4%。该法分析快速准确、灵敏度高、重现性好。A method using high-performance liquid chromatography(HPLC) coupled with photodiode array detector(PDA) was established for determination of astaxanthin.The separation was performed on Purospher STAR RP 18 column(4.6 mm×250 mm,5μm) with the mobile phase of methanol–water(Volume ratio was 95∶5).The detector was photodiode array detector and the variable wavelength detector(VWD) was set at 482 nm.The column temperature was 30℃ and the injection volume was 20 μL.The calibration curve was linear in the range of 0.2–16 μg/mL,and the correlation coefficient was 0.999 9.The relative standard deviation of determination results was 0.42%(n=6).The average recovery was 100.4%, and the detection limit was 0.01 μg/mL.This method is characteristic of rapidity,accuracy,high sensitivity and good reproducibility.海洋生物产业化中试技术研发公共服务平台项目(厦海渔合[2013]19号

Development of Glucosamine Sulfate Certified Reference Material

研制了硫酸氨基葡萄糖标准样品。以盐酸氨基葡萄糖为原料,制备高纯硫酸氨基葡萄糖,采用红外光谱(Ir)、高分辨质谱和核磁共振谱(nMr)进行结构确证。样品分装成200瓶样品后,采用高效液相色谱–蒸发光散射法进行均匀性检验、稳定性检验和定值分析。从样品中随机抽取15瓶进行均匀性检验,结果表明在95%的置信区间范围内样品均匀性良好。按照25℃长期试验稳定性(12个月)进行稳定性考察,结果表明在考察期间内样品稳定性良好。标准样品经国内8家具有分析资质的实验室进行协同定值,硫酸氨基葡萄糖标准样品定值结果为99.84%,相对扩展不确定度为0.18%(k=1.96)。该标准样品达到国家标准样品的技术要求,可用于有关硫酸氨基葡萄糖的分析方法校正和质量控制。Glucosamine sulfate certified reference material was developed.Glucosamine sulfate was made from the glucosamine hydrochloride.The structure of glucosamine sulfate certified reference material was comfirmed by infrared spectroscopy(IR), high resolution mass spectral and nuclear magnetic resonance(NMR).The sample was divided into 200 bottles, the homogeneity, stability testing and quantitative analysis were carried out by high performance liquid chromatography with evaporative light-scattering detector(HPLC–ELSD).According to analysis procedure of homogeneity under the confidence interval of 95%, 15 bottles of sample were randomly taken from 200 bottles, and the results were validated by F-test statistical methods.The stability inspection was carried on the long-term(12 months), and the results indicated that the period for glucosamine sulfate of storage was 12 months at 25℃.A cooperative certification was conducted with eight qualified laboratories.The certified purity value of the reference material of glucosamine sulfate was 99.84% with a relative expaned uncertainty of 0.18%(k=1.96).The reference material can conform to the technical requirement of the certified reference material.The material was intended for use in the method validation and quality control regarding glucosamine sulfate.厦门市科技计划项目(3502Z20130004); 海洋生物技术产业化中试技术研发公共服务平台(12PZP001SF10); 广东海洋经济发展区域示范项目(GD2012–D01–001

Determination of Eicosapentaenoic Acid Ethyl and Docosahexaenoic Acid Ethyl Ester in Fish Oil by Ultra Performance Liquid Chromatography

采用超高压液相方法检测鱼油中的二十碳五烯酸乙酯(EPA-EE)和二十二碳六烯酸乙酯(dHA-EE),采用uPlC bEH C18(2.1 MMx50 MM,1.7μM)色谱柱,考察了流动相有机相比例、流速及柱温对鱼油中EPA-EE和dHA-EE分离的影响,确定了最佳色谱条件:等度洗脱,流动相为乙腈-水(80∶20,体积比),流速0.5 Ml/MIn,柱温30℃,检测波长220 nM。该方法下,EPA-EE和dHA-EE分别在10.4μg/Ml~209μg/Ml、10.3μg/Ml~207μg/Ml范围内浓度和面积呈现良好的线性关系,相关系数r分别为0.999 4和0.999 7;EPA-EE和dHA-EE的方法的检测限为4 ng和6 ng,定量限分别为10 ng和15 ng,精密度试验rSd(n=6)分别为1.09%和0.34%,重复性试验rSd(n=6)分别为1.54%和1.25%,平均回收率分别为102.26%(rSd=1.33%)和96.64%(rSd=1.05%)。本方法快速、简单、可靠、灵敏、重复性好,可用于鱼油中EPA-EE和dHA-EE的快速检测。The article is researching determination of eicosapentaenoic acid ethyl ester(EPA-EE) and docosahexaenoic acid ethyl ester(DHA-EE) in Fish Oil by Ultra performance liquid chromatography(UPLC).The analysis was performed on an Acquity UPLC BEH C18 column(2.1 mm×50 mm,1.7 μm).The influence of flow rate,the proportion of organic phase in mobile phase,and column temperature on the separation of EPA-EE and DHA-EE in fish oil was comprehensively studied.The optimal separation condition was as follows: a mobile phase consisting of acetonitrile and water(80∶20,V/V) with a isocratic elution profile,UV detection wavelength at 220 nm,0.5 mL/min of flow rate for mobile phase,30 ℃ of column temperature.On the above condition,good linearity between response(peak area) and concentration was found over a concentration range of 10.4 μg/mL～209 μg/mL for EPA-EE,and 10.3 μg/mL～207 μg/mL for DHA-EE respectively.And the correlation coefficient of the calibration curve was 0.999 4 for EPA-EE,and 0.999 7 for DHA-EE respectively.The limits of detection(S/N>3) were 4ng for EPA-EE and 6ng for DHA-EE respectively.The limits of quantification(S/N>10) were 10ng for EPA-EE and 15ng for DHA-EE respectively.The average recovery rates of EPA-EE and DHA-EE were 102.26 %(RSD 1.33 %) and96.64 %(RSD 1.05 %) respectively.The RSD of repetition was 1.54 %(n=6) for EPA-EE and 1.25 %(n=6) for DHA-EE.This method was simple,accurate and sensitive with a good reproducibility.It is suitable for fast detection of EPA-EE and DHA-EE in fish oil.福建省科技计划重点项目(2009Y0035); 海洋公益性行业科研专项经费项目(200905022

Development of Trehalose Certified Reference Material

研制了海藻糖国家标准样品。以食品级海藻糖粗品为原料,纯化制备海藻糖,采用红外光谱(Ir)、质谱和核磁共振谱(nMr)以及单晶衍射等方法进行结构确证。样品分装成400瓶后,采用离子色谱法进行均匀性、稳定性检验和定值分析。从样品中随机抽取15瓶进行均匀性检验,经f检验表明,在95%的置信区间范围内,样品均匀性良好。在40℃下,经过24个月稳定性考察,结果表明样品稳定性良好。标准样品经国内8家具有分析资质的实验室进行协同定值,并评定了定值结果的不确定度,海藻糖标准样品定值结果为99.72%,扩展不确定度为0.26%(k=1.96)。该标准样品达到国家标准样品的技术要求,可用于有关海藻糖的方法校正和质量控制。Trehalose certified reference material was developed.Trehalose was purified from the crude product.The structure of trehalose certified reference material was comfirmed by infrared spectroscopy(IR),mass spectrometry,NRM and XRD.Trehalose was divided into 400 bottles.The homogeneity and stability testing and quantitative analysis were evaluated by ion chromatography method.According to analysis procedure of homogeneity,15 bottles of sample were randomly taken from 400 bottles,and the results were validated by F-test statistical methods.The stability inspection was carried on the long-term(24 months),and the results indicated that the period for trehalose storage was 24 months at 40 ℃.A cooperative certification was conducted by 8 qualified laboratories.The certified purity value of the reference material of trehalose was 99.72%with relative expaned uncertainty of 0.26%(k=1.96).The reference material can conform to the technical requirement of the certified reference material,and it can be used for validation and quality control regarding trehalose.福建省科技计划项目(2013N01010205); 厦门市海洋经济创新发展区域示范项目(12PZP001SF10); 海洋公益行业科研专项(201005020–1); 厦门市科技计划项目(3502Z20122009

Development of Squalene Certified Reference Material

为研制角鲨烯国家标准样品。以角鲨烯粗品为原料,制备高纯角鲨烯,采用红外光谱、高分辨质谱和核磁共振谱进行结构确证。样品分装成150瓶后,采用气相色谱-氢火焰离子化检测器法进行均匀性、稳定性检验和定值分析。从样品中随机抽取15瓶进行均匀性检验,经f检验表明在95%置信区间范围内样品均匀性良好;稳定性考察按照-18℃长期实验稳定性(24个月)进行,结果表明在考察期间内样品稳定性良好;标准样品经国内8家具有分析资质的实验室进行协同定值,并评定了定值结果的不确定度,角鲨烯标准样品定值结果为99.57%,相对扩展不确定度为0.30%(k=1.96)。该标准样品达到国家标准样品的技术要求,可用于有关角鲨烯的方法校正和质量控制。Squalene certified reference material was developed by purification of crude squalene.The structure of the certified reference material was confirmed by infrared spectroscopy(IR), high resolution mass spectrometry(HRMS) and nuclear magnetic resonance(NRM) and squalene was divided into 150 aliquots.The homogeneity and stability tests and quantitative analysis were carried out by gas chromatography-flame ionization detection(GC-FID).Fifteen randomly selected aliquots showed good homogeneity as indicated by F-test analysis at a 95% confidence interval.The test samples were stable at-18 ℃ for 24 months.A cooperative certification was conducted with 8 qualified laboratories.The certified purity value of the reference material was 99.57% with a relatively expanded uncertainty of 0.30%(k = 1.96).The reference material can conform to the technical requirement of the certified reference material.This material was intended for use in the method validation and quality control regarding squalene.厦门海洋研究开发院共建项目(2014); 海洋生物技术产业化中试技术研发公共服务平台项目(12PZP001SF10); 广东海洋经济发展区域示范项目(GD2012-D01-001

Development of Anhydrotetrodotoxin Certifi ed Reference Material

研制4,9-脱水河豚毒素国家标准样品。以河豚鱼卵巢为原料,提取制备4,9-脱水河豚毒素,采用红外光谱(Ir)、高分辨质谱和核磁共振谱(nMr)进行结构确证。样品分装成140瓶后,采用柱后衍生–高效液相荧光法进行均匀性、稳定性检验和定值分析。从样品中随机抽取15瓶进行均匀性检验,经f检验表明在95%的置信区间范围内样品均匀性良好;稳定性考察按照40℃加速试验稳定性(6个月)进行,结果表明在考察期间内样品稳定性良好;标准样品经国内8家具有分析资质的实验室进行协同定值,并评定了定值结果的不确定度,4,9-脱水河豚毒素标准样品定值结果为97.77%,相对扩展不确定度为0.4%(k=1.96)。该标准样品达到国家标准样品的技术要求,可用于有关脱水河豚毒素的方法校正和质量控制。4,9-anhydrotetrodotoxin certified reference material was developed.4,9-anhydrotetrodotoxin was made from the ovarium of pufferfish.The structure of anhydrotetrodotoxin certified reference material was comfirmed by mass spectrometry and NRM,infrared spectroscopy(IR).4,9-anhydrotetrodotoxin was divided into 140 bottles.The homogeneity and stability testing and quantitative analysis were evaluated by post column derivation high performance liquid chromatography–fl uorescent detection(HPLC–FLD) method.According to analysis procedure of homogeneity,15 bottles of sample were randomly taken from 140 bottles,and the results were validated by F-test statistical method.The stability inspection was carried on the short-term(6 months),and the results indicated that the period for anhydrotetrodotoxin of storage was 6 months at 40℃.A cooperative certifi cation was conducted with 8 qualifi ed laboratories.The certifi ed purity value of the reference material of 4,9-anhydrotetrodotoxin was 97.77% with relative expaned uncertainty of 0.40%(k=1.96).The reference material can conform to the technical requirement of the certified reference material.The material was intended for use in the method validation and quality control regarding anhydrontetrodotoxin.广东海洋经济发展区域示范项目(GD2012-D01-001); 海洋生物产业化中试技术研发公共服务平台(12PZP001SF10); 厦门市重大科技创新平台项目(3502Z20111001); 国家科技支撑计划(NO.2011BAK04B09

Optimization of degrease process during extracting collagen from sea-fish scales

研究了鱼鳞胶原蛋白提取过程中的脱脂工艺条件。采用l9(33)正交实验,考察了碱浓度、温度、时间等因素对鱼鳞脱脂工艺的影响,并使用离子色谱检测了脱脂溶液中羟脯氨酸的含量,以考察各种条件对鱼鳞胶原蛋白在脱脂工艺过程中溶出的影响。确定了鱼鳞胶原蛋白提取过程中的脱脂优化工艺,即温度18℃,碱浓度0.5MOl/l,时间8H。放大实验至原料2kg,得脱脂率为92%,适合规模化生产。Degreasation process during collagen extraction from sea-fish scales was explored.The effect of base concentration,temperature and time on degreasing were investigated with the orthogonal method.Ion chromatography was applied to observe the effects of factors on the dissolution of collagen from scales while degreasing.The optimum parameters were:temperature 18℃,base concentration 0.5mol/L and degreasing time 8h.The process was carried out at large scale well with degrease rate at 92%,which indicated that the process could be performed for plant-scale production.国家海洋局第三海洋研究所科研基本业务费(海三科2009006);国家科技支撑计划(2007BAB26B03

Determination of Domoic Acid in Human Plasma and Urine by High Performance Liquid Chromatography Tandem Mass Spectrometry

建立了高效液相色谱-串联三重四极杆质谱法(HPlC-MS/MS)测定人血浆及尿液中记忆丧失性贝毒软骨藻酸。人血浆及尿液样品加入6倍体积甲醇沉淀剂,涡旋离心后,取上清液进样测定。色谱柱为zOrbAX Sb C18柱(150x4.6MM,5μM),柱温30℃;流动相为乙腈-0.1%甲酸水溶液(13∶87,V/V),流速为1.0Ml/MIn。该方法检测血浆和尿液中软骨藻酸的线性范围均为1.8~115μg/l,相关系数r=0.999,检出限为0.9μg/l,定量下限为1.8μg/l。该方法具有操作方便、专属性强、灵敏度高的特点,适用于人体血浆及尿液中软骨藻酸的测定。High performance liquid chromatography(HPLC)combined with tandem mass spectrometry(LC/MS/MS)has been developed for the determination of domoic acid in human plasma and urine.6 times volume of methanol was added to the sample for protein precipipation;after vortex-centrifuging,the floated supernatant was injected into the HPLC-MS system.The separation was performed on a Zorbax SB C18(150×4.6mm,5μm),using acetonitrile and 0.1% formic acid in water(13∶87,V/V)as the mobile phase with the flowrate of 1.0mL/min.It was found that linear ranges in both plasma and urine samples were 1.8-115μg/L with the correlation coefficients of 0.999.The limit of detection(LOD)was 0.9μg/L and the limit of quantitation(LOQ)was 1.8μg/L.The method is simple,selective and sensitive for detection of domoic acid in human plasma and urine.国家973计划(No.2010CB428704); 国家自然科学基金(No.40831160519); 广东海洋经济发展区域示范项目(No.GD2012-D01-001

Structure identification and toxicity determination of 11-deoxytetrodotoxin

目的对制备获得的11-脱氧河豚毒素(11-dEOXyTTX)纯品进行结构确证与毒性测定。方法采用HrMS、1 H nMr和13 C nMr对样品进行结构表征;同时进行11-dEOXyTTX对小鼠的急性毒性试验。结果样品的HrMS图谱信息与11-dEOXyTTX相对分子质量吻合;1 H nMr和13 C nMr谱揭示了两组数据,该数据与文献报道基本一致;测得11-dEOXyTTX对小鼠的半数致死量(ld50)为(128.82±2.09)μg.kg-1。结论 11-dEOXyTTX是以半缩醛型-内酯型的互变异构体存在,与河豚毒素(TTX)的结构差别在于TTX中C-11上的羟基被氢取代。11-dEOXyTTX的毒性很强,但明显低于TTX的毒性,表明TTX中C-11上的羟基对阻滞钠离子进入钠通道也发挥了重要作用。Objective To identify the chemical structure and determine the acute toxicity of 11-deoxyTTX.Methods The sample was analyzed on the basis of HRMS,1H NMR and 13C NMR.Its acute toxicity was evaluated on mice.Results The mass spectrum was in very well agreement with the relative molecular mass of 11-deoxyTTX.The 1H NMR and 13C NMR spectra revealed double sets of signals,which were very similar to the literature.LD50 of 11-deoxyTTX to mice was(128.82±2.09) μg·kg-1.Conclusion 11-deoxyTTX exists as hemilactal-lactone tautomers.Its structural difference compared with TTX lies in the hydroxyl of TTX C-11 was replaced by hydrogen.11-deoxyTTX has a strong toxicity to mice,but it's remarkably lower than TTX.It indicated that the hydroxyl of TTX C-11 played an important role in the blockade of sodium channel.国家科技支撑计划项目(2007BAB26B02);基本科研业务费资助专项项目(海三科2008006