基于中空纤维的样品前处理技术及分离分析方法的开发与应用

发布时间：2018-04-21 15:43

本文选题：HF-LPME + 士的宁　；参考：《河北医科大学》2014年硕士论文

【摘要】：随着科学技术的迅猛发展，现代仪器分析的灵敏度、特异性已经大大提高，但在药物分析中，样品仍然需要进行适当的前处理，其占用的分析时间少则几分钟，有些甚至几十分钟或更长；样品的前处理时间可占整个分析时间的三分之二以上。因此，样品前处理技术在药物分析方法中有着举足轻重的作用。样品前处理技术的先进与否，直接关系到分析方法的优劣。发展省时高效、有机溶剂消耗少和集萃取、分离纯化、浓缩富集、进样于一体的新型样品前处理技术，已成为药物分析研究的热点领域之一。以中空纤维(Hollow Fiber，HF)为载体的样品前处理技术近几年有了飞快的发展。HF是一种有一定大小孔径、起分子筛作用的半透性膜形成的空心细管， HF膜具有选择透过性，可以使液体、气体混合物中某些组分从外向内腔或从内腔向外透过HF膜壁。HF优异的结构特点使其成为目前样品前处理技术研究的热点。中空纤维液相微萃取(Hollow Fiber Liquid-Phase Microextraction，HF-LPME)因其具有集分离纯化、富集浓缩、进样于一体的优点，在环境科学中具有广泛的应用。同样HF-LPME在药物分析中也得到了广泛的应用。随着人们对药品质量要求的提高，药品生产企业普遍采用了GMP管理模式，对企业设备清洁效率和管理提出了更高的要求。清洁后残留物分析的灵敏度是清洗验证的重要环节，是评价设备清洁效率的关键。本课题将HF-LPME作为设备清洗验证中分析方法的样品前处理技术，一步实现了样品的分离、纯化和富集，富集倍数达到两个数量级，极大地提高了分析方法的灵敏度，简化了实验操作，使得清洁验证的评价更加科学、可靠。 HF优异的超滤性能使其在生物制药、基因工程、血液透析等领域技术中广泛应用。HF对大分子物质的截留作用也使其在分析样品前处理中得到了迅猛发展。本课题利用中空纤维对大分子物质的截留作用，开发了一种从脂质体中分离未包封药物(即游离药物)的中空纤维离心超滤技术(Hollow Fiber Centrifugal Ultrafiltration，HF-CF-UF)，并将HF-CF-UF技术成功地应用于维生素A脂质体及难溶性药物两性霉素B脂质体的包封率测定。方法的分离过程基本保持了脂质体处方的原始的、稳定的存在环境(物理化学环境)，最大限度的减少了脂质体的泄露。课题比较了传统测定包封率的方法如凝胶色谱法(SEC)、固相萃取法(SPE)及离心超滤法(CF-UF)的方法特点。研究结果表明，HF-CF-UF不仅测定结果失真小，重现性好；而且操作简单，仅需一步离心即可有效的分离游离药物；而SEC、SPE不仅受到洗脱液物理化学环境与原脂质体基质不同，从而影响脂质体的稳定状态，且还受到洗脱液的断洗脱的影响，对脂质体的稳定状态影响较大，甚至导致游离药物浓度增加，测定结果失真。课题进一步探索了传统方法在测定脂质体包封率过程中出现的某些弊端的原因，为CF-UF分析表征脂质体包封率提供了理论依据。综上所述，以HF为载体的LPME的样品前处理技术不仅具有分离纯化功能，具有较高的富集效率，且操作简便、能有效避免交叉污染等突出优点，减少了繁杂操作带来的误差，特别适用于生产现场痕量组分的检测。以HF为载体的HF-CF-UF方法检测脂质体包封率，方法简单，分离过程保持了脂质体原始状态，方法失真小，且由于测定溶液没有稀释，测定误差小，重现性好。第一部分中空纤维液液微萃取技术在痕量药物分析中的研究与应用一中空纤维液液微萃取-HPLC法测定马钱子碱及士的宁清洁残留物目的：建立中空纤维两相液液微萃取(HF-LPME-HPLC)法，检测设备清洁验证中痕量士的宁和马钱子碱。方法：首先采用HF-LPME对样品进行纯化富集，其分离富集条件为：以正辛醇作为萃取溶剂，在800r/min搅拌速度下常温萃取60min。萃取后萃取溶液直接进样HPLC分析，色谱分析条件为：流动相为乙腈-0.4%磷酸水溶液(三乙胺调pH3.0)=13:87；流速1.0mL/min；检测波长260nm。结果：在优化的条件下，士的宁和马钱子碱富集倍数分别为120倍和80倍。士的宁在19.0ng/mL~1.90μg/mL之间线性关系良好(r2为0.9941)，最低检出限为2.98ng/mL，回收率为95.0%，RSD为5.1%；马钱子碱在11.0ng/mL~1.10μg/mL之间线性关系良好(r2为0.9948)，最低检出限为2.75ng/mL，回收率为98.6%，RSD为0.8%。结论：所建立的HF-LPME方法将样品中士的宁和马钱子碱的富集两个数量级，使其检测灵敏度大大提高，使清洁验证的评价更加科学、可靠。此外，该样品前处理方法操作简单快速、成本低、且环境友好，适用于生产现场清洁设备和容器中士的宁和马钱子碱残留的检测，为清洁验证评价中痕量药物残留的检测提供一种新的手段。二中空纤维液相微萃取-高效液相色谱法同时检测清洁过程中的4种痕量二氢吡啶类药物的残留目的：建立HF-LPME-HPLC法，用于设备清洁中4种二氢吡啶类药物(尼群地平、尼莫地平、费乐地平、间尼索地平)残留量的测定。方法：首先采用HF-LPME装置对样品进行纯化富集，萃取条件为：正辛醇为接受相，水为供给相，搅拌速度为800r/min，常温下萃取时间为80min。色谱分析条件采用Kromasil C18色谱柱(150mm×4.6mm，5μm)；流动相：乙腈-水(60:40，v/v)；流速：1.0mL/min；检测波长：237nm。结果：在优化的萃取条件下，尼群地平、尼莫地平、费乐地平的富集倍数达120倍左右，间尼索地平的富集倍数约为100倍。四种药物均在10~2.0×103μg/mL范围内线性关系良好，r2范围为0.9900～0.9982，平均回收率为93.0%～99.7%(RSD不高于4.0%)。结论：HF-LPME集分离纯化、浓缩富集、进样于一体，四种残留物的富集倍数均达到两个数量级，极大地提高了四种残留物分析方法的灵敏度。同时HF-LPME技术极大地简化了试验操作；且有机溶剂用量少，环境友好，可以用于企业的生产现场检测，为清洁验证过程中二氢吡啶类药物的痕量检测提供一种新的手段。第二部分中空纤维离心超滤法在脂质体质量表征中的应用一中空纤维离心超滤-HPLC法测定注射用两性霉素B脂质体的包封率目的：建立HF-CF-UF分离脂质体中未包封药物的前处理方法，并结合HPLC用于注射用两性霉素B脂质体的包封率的表征。方法：采用HF-CF-UF将未包封药物从脂质体中分离出来。取两性霉素B脂质体约0.2mL，置离心管中，将PSU膜材的中空纤维弯成U型后插入该管，置离心机中。在6000r/min条件下离心15min后打出超滤液直接注入色谱系统，测定未包封药物浓度。另取两性霉素B脂质体适量破乳后分析测定总药物浓度，并计算包封率。色谱分析采用Diamonsil C18色谱柱(250mm×4.6mm，5μm)，流动相为乙腈-0.02mol/L Na2EDTA(38:62)，流速：1.0mL/min，检测波长：405nm，柱温为常温。结果：HF-CF-UF方法仅需一步离心即可有效的将未包封的药物从脂质体中分离出来，且超滤液可以直接注入HPLC进行分析。两性霉素B在0.67～21.4μg/mL范围内线性关系良好(r2=0.9997)，回收率均在97%以上，RSD不大于2%。三批样品所测得的包封率分别为99.3%，99.6%，98.9%，RSD不大于1%。结论：本方法所用样品体积小，基本保持了脂质体处方的原始、稳定的存在环境(物理化学环境)，最大限度地减少了脂质体的泄露，结果准确且简便快速，为脂质体包封率的表征提供了一种新方法。二中空纤维离心超滤-HPLC法测定维生素A脂质体的包封率目的：建立HF-CF-UF分离脂质体中未包封药物的前处理方法，并结合HPLC法用于维生素A脂质体的包封率的表征。方法：取维生素A脂质体约0.2mL，置离心管中，将用维生素C棕榈酸酯溶液饱和的中空纤维弯成U型后插入该管，，置离心机中。在4000r/min条件下离心15min后打出超滤液直接注入色谱系统，测定未包封药物浓度。另取维生素A脂质体加甲醇破乳后分析测定总药物浓度，并计算包封率。色谱分析采用Diamonsil C18色谱柱(150mm×4.6mm，5μm)，流动相为纯甲醇，流速：1.0mL/min，检测波长：325nm。结果：试验结果表明，HF-CF-UF方法仅需一步离心即可有效的将未包封的药物从脂质体中分离出来，且超滤液可以直接注入HPLC进行分析。维生素A在0.258~8.24μg/mL范围内线性关系良好，回收率均在97.7%以上，RSD不大于1.5%。测得的三批维生素A脂质体包封率分别为98.8%，98.4%，98.9%，RSD不大于0.5%。结论：本方法操作简便，不破坏脂质体稳定的存在环境，克服了传统方法存在的某些弊端，结果准确，为脂质体包封率的表征提供了一种新方法。第三部分中空纤维离心超滤法在脂质体质量表征中的理论研究目的：将HF-CF-UF与传统的测定包封率的方法进行比较的基础上，提出了包封率测定过程中的动态平衡理论，并阐述了吸附现象的存在对难溶性药物制备的脂质体的包封率测定的影响，为脂质体质量表征时方法的选择提供了理论指导。方法：分别采用SEC、SPE、CF-UF及HF-CF-UF对维生素A脂质体和难溶性药物注射用两性霉素B脂质体进行包封率的测定。比较几种方法测得的差异，并进一步探索其原因。在SEC中，采用二次洗脱的方法，通过测定游离药物的浓度以阐述包封率测定结果偏低的原因。采用拆方分析法，测定脂质体处方中稳定剂在SPE柱的回收率，说明吸附现象对难溶性药物两性霉素B脂质体包封率测定结果的影响。结果：通过比较测定，SEC所测得的数值要比HF-CF-UF的数值低3%左右，通过进一步研究表明，SEC在测定过程中存在凝胶对脂质体的吸附现象及洗脱液导致脂质体渗漏的出现；选用SPE测定时，固定相填料的选择对测定具有重大影响，填料的疏水性作用易对AmB脂质体处方中的胆固醇和去氧胆酸钠产生吸附作用使得其不能被洗脱；而CF-UF中，除了浓差极化限制了其应用外，非特异性吸附也是不可忽视的。结论：HF-CF-UF避免了洗脱液的稀释作用，最大限度的保持了脂质体的存在状态，未包封的游离药物能自由穿过中空纤维膜，适合于脂质体中未包封的游离药物的分离测定。然而，传统的测定包封率的方法则操作繁琐费时，易受洗脱液、吸附现象的影响使得脂质体表征失真。
[Abstract]:With the rapid development of science and technology , the sensitivity and specificity of modern instrument analysis have been greatly improved , but in drug analysis , samples still need to be treated with proper pretreatment .
The pre - treatment time of the sample can account for more than two - thirds of the whole analysis time . Therefore , the pretreatment technology of the sample plays an important role in the drug analysis method . The advanced or not of the pretreatment technology of the sample is directly related to the advantages and disadvantages of the analysis method .

HF is a kind of hollow thin tube formed by semi - permeable membrane with pore size and molecular sieve function . The HF membrane has selective permeability .

HF - LPME ( HF - LPME ) has been widely used in the field of environmental science . The sensitivity of HF - LPME is the key to the cleaning efficiency of the equipment . The sensitivity of the residue analysis is two orders of magnitude , which greatly improves the sensitivity of the analytical method , simplifies the experiment operation , and makes the evaluation of cleaning verification more scientific and reliable .

HF - CF - UF ( HF - CF - UF ) has been successfully applied in the process of biological pharmacy , genetic engineering , hemodialysis and so on .
moreover , the operation is simple , and the free medicine can be effectively separated only by one - step centrifugation ;
In addition , the SEC and SPE are not only affected by the physicochemical environment of the eluent and the matrix of the original liposome , so that the stable state of the liposome is influenced , and the stability state of the liposome is greatly influenced by the elution of the eluent , and even the concentration of the free drug is increased , and the measurement result is distorted . The problem of the traditional method in determining the liposome encapsulation efficiency is further explored , and the theoretical basis for the CF - UF analysis to characterize the encapsulation efficiency of the liposome is provided .

In conclusion , the pre - treatment technology of LPME with HF as carrier not only has the advantages of separating and purifying , but also has the advantages of high enrichment efficiency , simple operation , effective avoiding cross contamination and the like , reduces the error caused by the complicated operation , and is especially suitable for the detection of trace components in the production site .

Study and application of the first part hollow fiber liquid - liquid micro - extraction technology in the analysis of trace drugs

Determination of the residues of brucine and sergeant by liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid - liquid -

Objective : To establish a hollow fiber two - phase liquid - liquid micro - extraction ( HF - LPME - HPLC ) method , and to test the determination of trace elements and brucine in equipment cleaning validation .

Methods : The samples were purified and enriched by HF - LPME . The separation and enrichment conditions were as follows : 1 - octanol was used as extraction solvent , and the extraction solution was extracted directly by HPLC . The chromatographic conditions were as follows : mobile phase consisted of acetonitrile - 0.4 % phosphoric acid aqueous solution ( triethylamine pH 3.0 ) = 13 : 87 ;
the flow velocity was 1.0 mL / min ;
The detection wavelength was 260 nm .

RESULTS : Under optimized conditions , the enrichment times of nine and brucine were 120 - fold and 80 - fold , respectively . The linear relationship was between 19.0ng / mL and 1.90渭g / mL ( r2 = 0.9941 ) , the lowest detection limit was 2.98ng / mL , the recovery rate was 95.0 % , RSD was 5.1 % ;
The linear relationship of brucine in 11.0 ng / mL ~ 1.10 渭g / mL was good ( r2 = 0.9948 ) , the lowest detection limit was 2.75 ng / mL , the recovery rate was 98 . 6 % , RSD was 0.8 % .

Conclusion : The established HF - LPME method has two orders of magnitude to enrich the sample sergeant and brucine , so that the detection sensitivity is greatly improved , so that the evaluation of the cleaning verification is more scientific and reliable . In addition , the pretreatment method is simple and rapid in operation , low in cost and environment - friendly , and is suitable for the detection of the residual of nine and brucine in the field cleaning equipment and the container , and provides a new means for the detection of trace drug residues in the cleaning verification evaluation .

Simultaneous detection of four trace amounts of dihydropyridines in the process of cleaning by liquid - phase micro - extraction - high performance liquid chromatography with high performance liquid chromatography

Objective : To establish an HF - LPME - HPLC method for determination of four kinds of dihydropyridines ( nitrendipine , nimodipine , felodipine and m - isoldipine ) in equipment cleaning .

Methods : The samples were purified and enriched with HF - LPME equipment . The extraction conditions were as follows : n - octanol as the receiving phase , water as supply phase , stirring speed of 800 r / min , extraction time of 80 min at room temperature . The chromatographic conditions were Kromasil C18 column ( 150mm 脳 4.6mm , 5渭m ) ;
Mobile phase : acetonitrile - water ( 60 : 40 , v / v ) ;
Flow rate : 1.0mL / min ;
Detection wavelength : 237nm .

RESULTS : Under the optimized extraction conditions , the enrichment times of nitrendipine , nimodipine and felodipine were about 120 times , and the enrichment times of internigroping were about 100 times . The linear relationship was good in the range of 10 ~ 2.0 脳 103 渭g / mL , r2 range was 0.9900 ~ 0.9982 , the average recovery was 93.0 % ~ 99.7 % ( RSD was not higher than 4.0 % ) .

Conclusion : The separation and purification , enrichment and injection of HF - LPME are two orders of magnitude , and the sensitivity of four residue analysis methods is greatly improved . The HF - LPME technology greatly simplifies the test operation .
and provides a new method for the trace detection of the dihydropyridines in the cleaning verification process .

Application of the second part hollow fiber centrifugal ultrafiltration method in the characterization of liposome quality

Determination of Encapsulation Rate of Amphioxins B liposomes for Injection by Hollow Fiber Centrifugal Ultra - filtration - HPLC

Objective : To establish a pre - treatment method for the unencapsulated drug in HF - CF - UF isolated liposomes , and to characterize the entrapment efficiency of the drug for injection by HPLC .

Methods : The unencapsulated drug was separated from liposome by HF - CF - UF . The hollow fibers of PSU membrane were inserted into the tube after centrifugation at 6000r / min . The concentration of unencapsulated drug was determined . After centrifugation for 15min at 6000r / min , the total drug concentration was determined and the entrapment efficiency was calculated . Diamonsil C18 column ( 250mm 脳 4.6mm , 5渭m ) was used as mobile phase . The mobile phase was acetonitrile - 0.02mol / L Na2EDTA ( 38 : 62 ) . The flow rate was 1.0mL / min , the detection wavelength was 405 nm , and the column temperature was normal temperature .

Results : The HF - CF - UF method can effectively separate the unencapsulated drug from the liposome by one - step centrifugation , and the ultrafiltrate can be directly injected into HPLC for analysis . The average recovery is more than 97 % and the RSD is not more than 2 % . The entrapment efficiency measured by the three samples is 99 . 3 % , 99.6 % , 98 . 9 % , and the RSD is not more than 1 % .

Conclusion : The sample volume used in this method is small , the original and stable existence environment ( physical and chemical environment ) of liposome prescription is maintained , the leakage of liposome is reduced to a maximum extent , the results are accurate and simple and rapid , and a new method is provided for the characterization of liposome entrapment efficiency .

Determination of encapsulation efficiency of vitamin A liposome by double - hollow fiber centrifugal ultrafiltration - HPLC

Objective : To establish a pretreatment method of non - encapsulated drug in HF - CF - UF isolated liposome , and to characterize the entrapment efficiency of vitamin A liposome by HPLC .

Methods : A liposome of vitamin A was put into centrifuge tube . The hollow fiber saturated with vitamin C palmitate solution was bent into U shape and inserted into the centrifuge . After centrifugation at 4000 r / min for 15min , the filtrate was injected directly into the chromatographic system . The concentration of unencapsulated drug was determined . The concentration of the unencapsulated drug was determined . The concentration of the total drug was determined after centrifugation at 4000 r / min . The concentration of the drug was determined . The chromatographic analysis was carried out with Diamonsil C18 column ( 150mm 脳 4.6mm , 5渭m ) . The mobile phase was pure methanol , the flow rate was 1.0mL / min , and the detection wavelength was 325nm .

Results : The results showed that the HF - CF - UF method can effectively separate the unencapsulated drug from the liposome by one - step centrifugation , and the ultrafiltrate can be directly injected into HPLC . The linear relationship of the vitamin A in the range of 0.258 - 8.24渭g / mL is good , the RSD is not more than 1.5 % . The entrapment efficiency of the three batches of vitamin A liposome is 98 . 8 % , 98 . 4 % , 98 . 9 % , and the RSD is not more than 0.5 % .

Conclusion : The method is simple and convenient to operate , does not destroy the stable existence environment of liposome , overcomes some defects existing in the traditional method , and provides a new method for the characterization of liposome encapsulation efficiency .

Theoretical study of the third part hollow fiber centrifugal ultrafiltration method in the characterization of liposome quality

Objective : Based on the comparison between HF - CF - UF and traditional method for determination of entrapment efficiency , the dynamic equilibrium theory in the measurement of entrapment efficiency was presented . The influence of adsorption phenomenon on the encapsulation rate of liposomes prepared by insoluble drug was discussed , and theoretical guidance was provided for the selection of the method of liposome quality characterization .

Methods : The encapsulation rate of vitamin A liposome and insoluble drug for injection was determined by SEC , SPE , CF - UF and HF - CF - UF . The reasons were compared .

Results : Compared with HF - CF - UF , the value of SEC was about 3 % lower than that of HF - CF - UF .
When the SPE is selected , the selection of the fixed phase filler has great influence on the measurement , and the hydrophobic effect of the filler is easy to generate an adsorption effect on the cholesterol and the sodium deoxycholate in the AmB liposome prescription so that the filler cannot be eluted ;
however , in CF - UF , non - specific adsorption is not negligible in addition to its application .

Conclusion : HF - CF - UF avoids the dilution effect of the eluent , maintains the existence state of the liposome , the unencapsulated free drug can freely pass through the hollow fiber membrane , is suitable for the separation and determination of the unencapsulated free drug in the liposome . However , the traditional method for measuring the encapsulation efficiency is cumbersome and time - consuming , is easy to be influenced by the eluent and the adsorption phenomenon , and the liposome is characterized by distortion .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917

【参考文献】