Dissertation
Dissertation > Medicine, health > Pharmacy > Drug analysis

Research and Application of Sample Pretreatment Methods Based on Hollow Fiber

Author XuMeng
Tutor JiangZuo
School Hebei Medical University
Course Pharmaceutical Analysis
Keywords HF-LPME Strychnine Brucine Dihydropyridine Cleaningvalidation HF-CF-UF SEC SPE CF-UF Entrapment efficiency Liposome Vitamin A Amphotericin B
CLC R917
Type Master's thesis
Year 2014
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In pharmaceutical analysis, increased sensitivity of analytical instrumentsand analysis of complicated object matrix put forward higher requirements forsample pretreatment technology. Therefore, sample pretreatment technologyplays a vital role in the development of pharmaceutical analysis. So thedevelopment of the new sample pretreatment technology is the main purposeof the scientists. The ideal sample pretreatment technology should meet withthe general requirements for high work efficiency, have good extraction andseparation efficiency. In addition it should set extraction, separation,purification, concentration enrichment sampling in one of the new samplepretreatment technique. Therefore, high sensitivity, quick and simple ways forsample pretreatment technology becomes a hot spot of research nowadays.The sample pretreatment based on hollow fibers as carriers developedrapidly in recent years. Hollow fibers are made of a certain pore size, themolecular sieve from the effects of thin semipermeable hollow tubes, whereinthe walls of the hollow fibers having selective permeability, the gas or theliquid mixture can pass from the cavity outwardly or from outside the lumenthrough the hollow fiber wall. At present, the sample pretreatment based onhollow fibers as carriers such as hollow fiber liquid phase microextraction(HF-LPME) has its advantages for holding a set of separation and purification,enrichment and concentrated, injection in one of the operation and have a widerange of applications in environmental science. The applications of HF-LPMEin pharmaceutical analysis have gradually widened at the same time. Thissubject applies HF-LPME in the production equipment cleaning validationsystem. For cleaning validation is a key factor in drug companies to ensuredrug quality. In cleaning validation, the sensitivity of the analysis method is ofgreat importance to the effectiveness of cleaning and to prevent cross-contamination. This topic will adopt HF-LPME as equipment cleaningvalidation sample pretreatment technology prior to the analysis with object toachieve the separation, purification and enrichment of the sample. TheHF-LPME will greatly improve the sensitivity of the analysis methods. What’smore, the simplified experimental operation makes the cleaning validationprocess more scientific and sensitive.In addition, the porous and the interception hollow fibers made it also hasa wide range in ultrafiltration applications, such as: hemodialysis, desalination,gas separation, sewage treatment and other fields. This object adopts theintercept of hollow fiber to develop a new centrifugal ultrafiltrationtechnology: hollow fiber centrifugal ultrafiltration (CF-UF' dissertation">HF-CF-UF) and applyHF-CF-UF innovatively in the analysis of encapsulation efficiency in vitaminA liposome and insoluble drugs amphotericin B liposome. The results showedthat HF-CF-UF has tremendous advantage than the traditional methods for thereasons below: a small sample volume, easy to operate, hold the existence of astable liposome environment, accurate analysis of liposome encapsulation rate.In addition, the subject made a comparative study comparing this method withthe traditional methods such as SEC, SPE, CF-UF. Based on this, weestablished a theoretical study in the analysis of the entrapment efficiency ofliposomes. Therefore, this study provides a new method for thecharacterization of the encapsulation efficiency of liposomes and makes thecharacterization of the liposome quality more scientific and reasonable.In summary, the HF-LPME and HF-CF-UF sample pretreatmenttechnology has the advantages of low cost, fast, easy, simple device, avoidingcross-contamination and so on. What’s more, they can not only reduce staffdue to different operating and sample error caused by multiple transfers, butalso avoid the use of large amounts of organic solvents and reduceenvironmental pollution. Therefore, the sample pretreatment technology basedon hollow fiber has good prospects in the future and is worth attentionresearch. PART1Study and application of HF-LPME as a sample pretreatmentmethod in trace drug analysis1Determination of Strychnine and Brucine in Cleaning Residue by HollowFiber-Liquid Phase Microextraction coupled with HPLCObjective: A hollow fiber-liquid phase microextraction(HF-LPME)-HPLC method was established for the determination of tracestrychnine and brucine in cleaning validation of productional equipment.Methods: The experimental parameters of HF-LPME were controlledand optimized: the extraction solvent only was100μL n-octyl alcohol, theextraction procedure was finished at a stirring rate of800r/min for1h. AnHPLC method was applied for the determination. A C18column was used withthe mobile phase of0.4%phosphoric acid (adjusted to pH3.0bytriethylamine)-acetonitrile (85:15) at the detection wavelength of260nm.Results: Parameters influencing the extraction efficiency wereinvestigated and optimized. The enrichment factors of strychnine and brucinewere80.6and122.4, respectively. Strychnine showed a good linearrelationship at a range of19.0ng/ml~1.90μg/mL (r2=0.9941) with the limit ofdetection of2.98ng/mL and average recovery of95.0%(RSD=5.1%). Brucineshowed a good linear relationship at a range of11.0ng/mL~1.10μg/mL(r2=0.9948) with the limit of detection of2.75ng/mL and average recovery of98.6%(RSD=0.8%).The calibration curves for strychnine and brucine werelinear in the range of0.01~1μg/mL. Their recoveries were98.3%and98.5%,with RSDs of0.4%and0.6%.Conclusion: The detection sensitivity was greatly improved by usingHF-LPME. Moreover, the method is simple, low cost, environmental friendly,therefore it was suitable for the suitable for the determination of strychnineand brucine in cleaning validation.2Simultaneous determination of four different trace dihydropyridines oncleaning validation by hollow fiber liquid microextraction coupled with HPLCObjective: An HPLC method with hollow fiber liquid microextraction (HF-LPME) was established for simultaneous determination of four differenttrace dihydropyridines (Nitrendipine, Nimodipine, Felodipine andm-Nisoldipine) on cleaning validation.Methods: With homemade HF-LPME device, the extraction conditionwas optimized. The HF-LPME used1-octanol as acceptor phase and aqueoussolution as donor phase. The extraction procedure was finished at800r/minfor80min. The chromatographic conditions used Kromasil C18column. Themobile phase consisted of acetonitrile-water (60:40). The detectivewavelength was237nm. Flow rate is1.0mL/min.Results: Under the optimal extraction condition the enrichment factors ofnitrendipine, nimodipine and felodipine were about120, while m-nisoldipineabout100. The analytes showed a good linear relationship in the range10~2.0×103μg/mL (r2=0.9900~0.9982). The average recoveries ranged from93.0%to99.7%(RSD≤4.0%).Conclusion: The method is simple, rapid and very low organic solventconsumption and high enriching times. It has a great significance on theanalysis of trace dihydropyridines on cleaning validation.Part2Application of HF-CF-UF in quality characterization of liposomes1Determination of the entrapment efficiency of amphotericin B liposome byhollow fiber centrifugal ultrafiltration coupled with HPLCObjective: In the present study, a robust, specific and sensitive methodhas been developed to effectively separate the unencapsulated drug andliposomal drug under equilibrium conditions by the hollow fiber centrifugalultrafiltration (HF-CF-UF).Methods: Briefly,0.2mL sample was placed into a centrifugation device,and then a15-cm piece of hollow fiber was inserted into the glass tube. Thefiltrate containing free drug was obtained by HF-CF-UF and analyzed byHPLC through direct injection. Amphotericin B was monitored at a UVwavelength of405nm and separation was accomplished on a Diamonsil C18column (250×4.6mm,5μm; Dikma) at room temperature. The mobile phase was acetonitrile-0.02mol/L Na2EDTA (38:62), while the flow rate was set at1mL/min.Results: Amphotericin B was found to be linear in the concentration0.67~21.4μg/mL (r2=0.9997). The average recovery was better than97%withthe RSD less than2.0. The LOD and LOQ were67and218ng/mL,respectively. The entrapment efficiency of the threes batches samples were99.3%,99.6%,98.9%with RSD less than1.0%.Conclusion: The method provided a more reliable separation of free andliposomal drug without yielding artificially high free drug concentrations. Itprovides a simple and fast ultrafiltration means for the analysis of theentrapment efficiency of liposomes. Since the method kept the original state ofsample, it may be developed as an ideal method for evaluation of theentrapment efficiency of the liposomes.2Determination of the entrapment efficiency of liposome Vitamin A byhollow fiber centrifugal ultrafiltration coupled with HPLCObjective: In the present study, a robust, specific and sensitive methodhas been developed to effectively separate the unencapsulated drug andliposomal drug under equilibrium conditions by the hollow fiber centrifugalultrafiltration (HF-CF-UF).Methods: Briefly,0.2mL sample was placed into a centrifugation device,and then a15-cm piece of hollow fiber was inserted into the glass tube. Thefiltrate containing free drug was obtained by HF-CF-UF and analyzed byHPLC through direct injection. Vitamin A was monitored at a UV wavelengthof325nm and separation was accomplished on a Diamonsil C18column(150×4.6mm,5μm; Dikma) at room temperature. The mobile phase wasmethanol (100%), while the flow rate was set at1mL/min.Results: Vitamin A was found to be linear in the concentration0.258~8.24μg/mL. The average recovery was better than97.7%with the RSDless than1.5. The LOD and LOQ were51and165ng/mL, respectively. Theentrapment efficiency of the threes batches samples were98.8%,98.4%,98.9%with RSD less than0.5%. Conclusion: The HF-CF-UF technique proved to have severaladvantages compared to other previously reported extractiontechniques-namely, more accurate without affecting the balance of liposome,convenient and simple and lower detection limits. Moreover, the techniquerequires very small samples, which is suitable for analysis of liposomes.Recovery was notably high and the performance of the proposed methodologywas most satisfactory. Therefore, it can replace other techniques like the lessprecise SEC or the complex SPE.Part3Theoretical study of HF-CF-UF in quality characterization ofliposomesObjective: Further investigation into the methods for analysis of theentrapment efficiency of liposomes has been prompted by the urgent need toimprove traditional methods which offer more risk to liposome leakage. Basedon the comparision with previously developed nonequilibrium procedures, wehave developed and characterized an equilibrium method of separating freeand liposomal drug for liposomal formulations. Morever, we investigated theeffect of adsorption phenomenon on the analysis of the entrapment efficiencyof AmB liposome. The study provides a theoretical guidance for the analysisof the entrapment efficiency of liposomes.Method: We compared the entrapment efficiency of two liposomes: ie:vitamin A liposome and amphotericin B liposome using SEC、SPE、CF-UFand HF-CF-UF, respectively. In addition, we investigated the reasons of thepossible problems leading to the errors.Results: Evaluation of the entrapped fraction with SEC resulted in3percent or4percent lower entrapment values. In addition, further investigationdata suggested that SEC could lead to liposome leakage and adsorption oflipids on the column gel exited during the measurement. For SPE, the SPEcolumns which had a great hydrophobic stationary phase could adsorbliposomes, thus causing the liposomes can not be eluted. Method like CF-UFsuffered from NSB. Conclusion: HF-CF-UF avoids dilution of the eluate and holding thestate of liposomes. The small concentration of free drug can pass through thehollow fiber membranes which is suitable for the measurement of theentrapment efficiency of liposomes. However, the traditional methods sufferedfrom some defects, for example: yielding high free drug concentration,complicated extraction system and procedure, long analysis time, et al. SinceHF-CF-UF kept the original state of samples, it may be developed as an idealmethod in evaluation of the entrapment efficiency of the liposomes.

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