Research And Application Of New Sample Processing Methods In Pharmaceutical Analysis

In pharmaceutical analysis, the sample treatment plays a key role in the whole analytical process, the main purpose is to extract the analysis from its matrix, isolation, purification and hold concurrently at the enrichment of function, so as to improve the accuracy, specificity and sensitivity. The ideal analysis method of sample with the general requirements for high work efficiency, have good extraction and separation efficiency, hold concurrently purification effects and simple operation, so it reduces less because of multistep processing and appears with the pollution losses. Therefore, high sensitivity, quick and simple ways for sample treatments become a hot spot of research nowadays.At present, the most common sample processing methods are offline methods, such as solvent extraction, ultrasonic extraction and protein precipitation of traditional technology. Micro extraction technology with high selectivity guarantees the good reproducibility as an effective analysis sample pretreatment method, develop fast in recent years. The way for processing mainly has solid phase micro extraction and liquid phase micro extraction; in addition, micro dialysis and ultrafiltration method of such technology are also the most common methods.In pharmaceutical analysis, especially in biological drugs analysis, the separation or removal of macromolecule, often are the main purpose and key steps of the sample pretreatment. It not only influences the accuracy and precision of the analysis results, and the key is restricted by the work efficiency. At present, commonly used methods were mainly with all kinds of precipitation centrifugation, gel osmosis method and technique etc, however these technology tends to exist some shortcomings such as complex, time-consuming and operational steps cost higher. Ultrafiltration technology is a relatively simple sample pretreatment method, but the membrane in the traditional ultra filtration device is generally flat membrane form, the device in the ultra filtration process can make a strong pharmaceutical analysis, indirectly leads to film seepage flow down and separation efficiency reduce, filtrate difficult to collect and expensive cost analysis, also make it difficult to popularize. This subject adopts a new kind of hollow fiber centrifugal ultra filtration technology, in centrifugal ultrafiltration, centrifugal and hollow fiber membrane are parallel, the centrifugal force eliminates the membrane surface strong bad, it simplifies the polarization phenomenons, avoids the dilution effect, also reduced the cost analysis.Another important task of the sample pretreatment is to analyze the target with concentration, to improve the concentration of the sensitivity analysis method, especially for improving the sensitivity of some analysis techniques which is difficult to improve from hardware, such as capillary electrophoresis. The traditional enrichment methods are mainly using liquid-liquid extraction and total precipitation etc. In recent years, solid phase micro extraction (SPE), all kinds of liquid-liquid micro extraction technology (LPME) and the empty enrichment technology vigorous development, to make up for the efficiency of traditional preparation technology. However, these techniques all belong to offline separation, enrichment technology, its operation procedure is tedious, time-consuming and higher shortage cost. So, online research and application of enrichment technique get people's increasing attention.Capillary electrophoresis (CE) is an efficient separation technology which was developed in the 1980s, however, because of its tiny into injection volume and shorter testing process, makes the light of trace and trace components in the CE separation of analysis severely constrained. Although people can improve high-sensitivity through the use of special designs testing pool or application high-sensitivity detector, but the instrument prices and expensive and the special requirements of some sample properties, limiting its application and popularization. A more feasible and more reasonable solution is to sample (or in online column) enrichment. This method is based on the sample, background buffer solution composition and into injection procedures simple regulation and realizing of reconstruction, no need to transform the instrument.Capillary electrophoresis field amplification enrichment technology is a kind of capillary online enrichment technique. Its principle is using electricity migration, and adds the low concentration into sample to sample solution of liquid filled with high concentrations of buffer of capillary. When the electrical migration into the sample, the electric field strength injector is much higher than the electric intensity, capillaries in the injector which has high samples ion will electrophoresis migration velocity; On the other hand, the background of electrolyte solution electrophoresis migration velocity changed, so that the basic ion can faster migration to background electrolyte solution. Sample ion in the sample solution and background electrolyte concentration happened get border piling up.In this topic, online capillary field amplification was used to analyze the effective components of trace toxicity aconitine, biological samples as well as the trace compounds in pharmaceutical preparations, it provides a reliable method for the quality control of pharmaceutical preparations.Part 1 The application of new HF-CF-UF device in the sample pretreatment(1) To determine the entrapment efficiency in the Indomethacin liposome by hollow-fiber ultrafiltration then centrifugation for HPLC analysisObjective: Determination of unenveloped micromolecule drugs in liposome by using the new HF-CF-UF device, so as to calculate the entrapment efficiency in the Indomethacin liposome.Methods: The essay was infused into a centrifugation wipe at 0.2 mL, then a piece of 15 cm hollow fiber was inserted into the pipe. After the centrifugation lasting for 20 min, the filtrate was imbibed. Then the filtrate was injected into the HPLC system. The concentration of the free drug was applied according to the chromatographic conditions below. Then the liposome was solved with the methanol, the concentration of the total drug could be applied according to the chromatographic conditions below, the entrapment efficiency was calculated by the data above. The chromatographic condition would be as follows, a Hypersil C18 column (4.6×150 mm, 5μm), mobile phase: 0.1 mol/L acetic acid solution-Acetonitriles (v/v=45:55), at the flow rate of 1.0 mL·min-1, the detection of the UV wavelength was set at 320nm.Results: The free indomethacin was well separated from the essay and other giant molecules by the ultrafiltration via hollow fiber. The filtrate could be directly injected into the HPLC system. Besides the recovery to filter with the free drug and the total drug was more than 97.0% and 97.5%, and the RSD was less than 1.0%(n=5).Conclusion: The method is simple and rapid by using the new HF-CF-UF device, therefore it may be used as a new medium for the quality control of indomethacin liposomes.(2) Pretreatment of plasma samples by a novel hollow fiber centrifugal ultrafiltrate device for the determination of cefaclor concentrations in human plasmaObjective: to establish a method by using a novel hollow fiber centrifugal ultrafiltrate device to remove the macromolecule in the plasma, study the influence between the free drug and protein binding and determine the blood drug level of cefaclor.Methods: Adjusted plasma sample to acidity,it was transferred into centrifuge tube within a U shape hollow fiber. After centrifugalization for 20min, the filtrate could be directly injected into the HPLC system. A Diamonsil C18 column was used. The mobile phase consisted of acetonitrile- tetrahydrofuran-10mmol sodium dihydrogen phosphate (pH 2.9)(6:5:89). The detective wavelength was 265 nm. Cefradine was used as internal standard. Flow rate is 1.0 mL/min.Results: When the pH is 2, the bond drugs can free from the plasma protein. The average extraction recovery was no less than 86.8%. The absolute recovery was no less than 99.2%. The linearity was obtained from 0.06 to 30.72μg·mL-1, and the detective limit of concentration was 0.02μg·mL-1. The inter-day RSD and intra-day RSD were less than 1.8% and 3.6%, respectively.Conclusion: A novel hollow fiber centrifugal ultrafiltrate device was applied in the proposed method, only one step was taken in the whole pretreatment process, it was simple and fast. Sample diluting was avoided to improve the sensitivity. The new method refers to the quantitative analysis of cefaclor in human plasma.Part 2 Determination of aconitine and hypaconitine in Gucixiaotongye by capillary electrophoresis with field-amplified sample injectionObjective: To set up a capillary electrophoresis with field-amplified sample injection method for the determination of aconitine and hypaconitine in Gucixiaotongye.Methods: Samples were treatment by HCl, first, then removed the impurity by ether. After alkalify and extracted by ether, removed ether and resolved by 10% acetonitrile. An uncoated fused-silica capillary column(50 m×50 cm, effective length 42 cm) was used. The capillary inlet was dipped in methanol for 5s prior to electrokinetic injection 30 s, the injection voltage is 12KV. The running buffer was 50 mmol·L-1 phosphate electrolyte solution (pH9)-methanol (90: 10). The running voltage was 10 kV and the detection wavelength was 235 nm.Results: The enrichment factor is 500 for aconitine alkaloid with this method. Aconitine and hypaconitine were found to be linear in the concentration 17.2~275 ng·mL-1 and 34.4~550 ng·mL-1, the average recovery was better than 93.9% with the RSD of 3.8%.Conclusion: The method is simple, rapid and specific with high stacking efficiency, it provides a new reliable means for production and quality control of Gucixiaotongye.