The Application Of Mini-centrifugal Ultrafiltration Device In Sample Pretreatment For Pharmaceutical Analysis

It is very important for the content evaluation of active compound in pharmaceutical analysis. It is difficult to separate micromolecule if they are combination or coexistence with macromolecule, so that the preparation of the sample is the key procedure in the research work in that cases.Precipitant or extraction with organic solvent were frequently used to separate the analyte from the sample matrix in pharmaceutical analysis. However, the traditional pretreatment and enrichment methods are laborious and time consuming, furthermore the residual macromolecule may be adsorbed on the packing, which may shorten the life of the column. High efficiency purification of analyte was hardly obtained by traditionally phase separation method if the similar polarity of micromolecule were combined or coexisted with macromolecule in sample matrix. Sample pretreatment procedure is known to have a significance effect on accuracy and reproducibility. Macromolecule is needed to separate first in most sample pretreatment, in which the step is numerous, and the cost is expensive.The presence environment of biological matrix may have changed as the addition of extraction solvents and precipitation agents in analyzing micromolecule of biological samples. Since the original drug binding state have changed, therefore it is unable to obtain the free drug concentration. So that it is usually monitor the total drug concentration in biological samples, which is the main task of therapeutic drug monitoring. Keeping the original environment of biological matrix and the combination of drug in biological matrix are key procedures in evaluating of free drug concentration.Ultrafiltration was widely used in large scale sample preparation and biomedicine purification, which could isolated micromolecule from a complex matrix samples. But in analitycal scale, only a few reports mentioned mini-ultrafitration device, in which the membrane was flat placed in the centrifuge tube. The concentration polarization appeared on the flat membrane, which reducing the amount of seepage flow and separation efficiency. And filtrate was difficult to collect, in addition, expensive analysis cost limited to its application. The aim of present project is to design a simple, cheap and mini-centrifuge ultrafiltration device for the sample pretreatment.The purpose of present research is to isolate the target analyte effectively from the complex matrix samples, which contain macromolecule and micromolecule in pharmaceutical analysis by using mini-centrifugal ultrafiltration device, based on hollow fiber membrane as the separated media. In the proposed device, the membrane of hollow fiber was parallel with the centrifugal force, so the concentration polarization was eliminated. Compared with traditional methods, the developed method is time saving, labor saving and inexpensive. Phase transition that frequently happened in protein precipitation method or organic solvent extraction method, resulted in precipitation adsorption. But, there was no phase transition in the proposed method and so the precipitation adsorption is reduced. The device used in present work can be not only used to remove macromolecule in pharmaceutical analysis, but also to analyze micromolecule in biological fluids. The filtrate obtained from the proposed device can be directly inject to chromatographic system without dilution and disrupting the physiological state of the sample. The establishment of the proposed method providing an effective sample pretreatment technique to analysis the free drug concentration, which will reflected the real relationship between drug concentration and pharmacological strength. It is also provide a novel mean for the quality control of pharmaceutical products.PART 1 Evaluation of micromolecule in pharmaceutical analysis by hollow fiber centrifugal ultrafiltration(1) Determination of maltose in Human Normal Immunoglobulin for intravenous use by hollow fiber centrifugal ultrafiltration-HPLC Objective:To use of hollow fiber centrifugal ultrafiltration for fast separation of maltose in Human Normal Immunoglobulin for intravenous use, and determine its content by HPLC.Methods:About 10 mL sample solution was put into hollow fiber centrifugal ultrafiltration device, which was centrifuged at 1.25×103 g for 15 min. Then the filtrate was withdraw from the hollow fiber into syringe and 20μL filtrate was directly injected into the HPLC for analysis. The separation was carried out on an Inertsil NH2 column (250 mm×4.6 mm,5μm) with acetonitrile-water(70:30) as the mobile phase at a flow rate of 1.0 mL·min-1. The detector was refractometer and the column temperature was 40℃.Results:After centrifugal ultrafiltration, there was no protein interference in filtrate, which can be injected directly into chromatography. The method can be used for sample pretreatment of maltose in Human Normal Immunoglobulin for intravenous use, which is simple and rapid. The recovery was good, and the average recovery was 99.9% with the RSD of 1.4%. A good linear relation was obtained in the range of 1.00 mg·mL-1～5.00 mg·mL-1 (r=0.9999).Conclusion:Hollow fiber centrifugal ultrafiltration method overcomes concentration polarization and adsorption. Compared with traditional method of protein precipitation, there was no interference of macromolecules protein in the filtrate after centrifugal, which made the results more accurate and extended the column life. It provides a simple and cheap ultrafiltration means for the analysis of micro sample and it is also suitable for thr pretreatment of micromolecule in globulin products.(2) Determination of benzalkonium bromide in Hypromellose Eye Drops by hollow fiber centrifugal ultrafiltration-HPLC Objective:To establish a simple method for the determination of benzalkonium bromide by HPLC. Using of hollow fiber centrifugal ultrafiltration method in Hypromellose eye drops for sample pretreatment, in order to remove macromolecules hypromellose, which will damage the chromatographic system.Methods:The sample placed in hollow fiber ultrafiltration device, was centrifuged at 1.25×103 g for 15 min. So, a good separation was obtained between macromolecule hypromellose and micromolecule benzalkonium bromide. Then the filtrate was withdraw from the hollow fiber into syringe and 20μL filtrate was injected directly into the HPLC for analysis. The separation was carried out on a Hypersil C18 column (250mm×4.6mm,5μm) with acetonitrile-70mmol·L-1 ammonium acetate consisting of 1% triethylamine, adjusted with acetic acid to pH 5.0 (70:30) as the mobile phase at a flow rate of 1.OmL·min-1 with detection at 262 nm.Results:Enough filtrate was obtained in 15 min by using hollow fiber centrifugal filtration. It was also elemented the damage of hypromellose to the chromatographic system, and extending the column life. The average recovery was 99.6% with the RSD of 1.1%. A good linear relation was obtained in the range of 12.5μg·mL-1～200μg·mL-1 (r=0.9999).Conclusion:After a simple centrifugation, micromolecule benzalkonium bromide was separated from the sample in 15 min. There was no damage of macromolecule hypromellose to the chromatographic system, and the hollow fiber can be repeatedly used, which reduced the cost of analysis. It also provides a new, convenient and practical analytic means for the separation and analysis of benzalkonium bromide.(3) Simple approach for determination of acetyltryptophan in Human albumin by direct injection using HPLC followed by hollow fiber centrifugal ultrafiltrationObjective:A sample pretreatment method was established for analysis acetyltryptophan in human albumin by using hollow fiber ultrafiltration, and determined the content of acetyltryptophan by HPLC.Methods:About 10 mL sample solution, which has already diluted 100-fold by water, and added 100μL 0.1% phosphate, was put into hollow fiber centrifugal ultrafiltration device with centrifgual force for about 15 min. Hollow fiber was saturated with glycine before use. The filtrate was withdraw from the hollow fiber into syringe and 20μL filtrate was injected directly into the HPLC for analysis. The separation was carried out on a Hypersil C18 column (250 mm×4.6 mm, 5μm) with methanol-water (45:55) (consisting of 0.1%phosphoric acid) as the mobile phase at a flow rate of 1.0 mL·min-1 with detection at 280 nm.Results:Acetyltryptophan can be separated from human albumin by using hollow fiber saturated with glycine centrifugal ultrafiltration device. The filtrate within the hollow fiber is easy to be collected after centrifugation, which can be injected directly into chromatography. It can overcome adsorption phenomenon by using hollow fiber saturated with glycine. A good linear relation was obtained in the range of 10.0μg·mL-1～160μg·mL-1 (r=0.9999) and the average recovery was 99.4% with the RSD of 0.8%.Conclusion:It is easy to exclude the impact of protein to acetyltryptophan by using hollow fiber centrifugal ultrafiltration, and it is suitable for the quality control of Human albumin in production processPART 2 Determination of free concentration of carbamazepine by hollow fiber centrifugal ultrafiltration-HPLC in plasmaObjective:A novel, simple and fast sample pretreatment was developed for the analysis of free carbamazepine concentration in plasma. Methods:The 0.2 mL plasma without dilution was put into home made hollow fiber ultrafiltration device with relative centrifgual force of 9.59×102 g. Then the filtrate was withdraw from the hollow fiber into syringe and 20μL filtrate was injected directly into the HPLC for analysis. The separation was carried out on a ZORBAX SB-C18 column (250 mm×4.6 mm,5μm) with methanol-water(52:48) as the mobile phase at a flow rate of 1.0 mL·min-1 with detection at 254 nm.Results:Human plasma samples were put into the hollow fiber centrifuge centrifugation, and after centrifugation, the concentration of carbamazepine in filtrate and the free concentration of carbamazepine in plasma were equal. Then the filtrate was directly into the HPLC for analysis. A good linear relation was obtained in the range of 0.624μg·mL-1～20.0μg·mL-1 (r=0.9991) and the average recovery was 99.5% with the RSD of 1.2 %. Compared with 10 patients by the total drug concentration and free drug concentration.Conclusion:Without destroying the sample in physiological state, free drug and protein binding can be separated by using Hollow fiber centrifugal ultrafiltration technology, and then the free drug concentration in plasma was obtained. Analysis result showed that 10 patients in the normal physiological state conditions, the total concentration and free concentration of carbamazepine are basic correlated, but under certain conditions, especially in pathological conditions, the concentration of free drug may have a greater change. Therefore, monitoring free drug plays an important role for the safety and usage.