Study And Application Of Sample Pretreatment Of Volatile Components In Pharmaceuticals

Sample pretreatment before instrumental analysis is usually necessary for the analysis of volatile components in pharmaceuticals in order to separate, purify, and enrich these components. It can decrease the interference to the analytes and have no contaimination for chromatography system and so on. Traditional extraction procedures such as volatile oil extractor extraction, hydrodistillation, ultrasound extraction, soxshlet extraction etc. which usually time-consuming and labor intensive, which requires large amounts of toxic and environmentally unfriendly organic solvents. In addition, these traditional extractions are mostly performed using the open system, which leads to the loss of volatile components and causes the inaccuracy of the results. Recently, solid-phase extraction and solid-phase microextraction have already been applied to determin volatile components in pharmaceticals. But the materials of extraction device coatings are relatively few, expensive, short life and the carrying-over effects are usually inevitable. Therefore, it is essential to develop rapid, accurate, high selectivity, airtight and environmentally friendly sample pretreatment methods for the analysis of volatile components in pharmaceuticals.Nowadays, headspace technology, with its simple installation, low cost, less pollution on the chromatographic system, fast analysis, and sensitivity characteristics has more and more applied for the determination of organic residual solvents. Rational selection of headspace matrix media is the key in the headspace gas chromatography (HS-GC). An ideal headspace matrix medium should not only dissolve the pharmaceticals and analytes, but also have good headspace efficiency, that is, high sensitivity. Water is a traditional matrix medium and the most ideal one. Therefore, in this study water as matrix medium for the determination of residual solvent ethanol in Huoxiang Zhengqi oral liquid by HS-GC was estabilished firstly. The reason to choose water as matrix medium is that Huoxiang Zhengqi oral liquid and the analyted ethanol are both soluble in water, and water is environmentally friendly. Moreover, the wide chromatographic peak which may be produced by DMF, DMSO, etc. is avoided. The proposed method is proved to be simple, accurate, and sensitive. It not only decreases the interference to the analytes but aslo has no contaimination for GC system, which is suitable for the determination of residual solvent ethanol in Huoxiang Zhengqi oral liquid.Water is the ideal matrix medium due to the hydrophobic effect for water-insoluble components and shows good headspace efficiency. However, for water-insoluble drugs and analytes, the traditional matrix media such as DMA, DMSO et al can produce wide chromatographic peak in the process of analysis that interfere with analytes, even contaminate the detection system. If the problems of dissolution of water-insoluble drugs and analytes are avioded, water can be used as ideal matrix medium in the analysis of more pharmaceticals. In this paper, by adding the modifier—methanol in matrix medium—water, a method for the determination of menthol and methyl salicylate in Baicao oils by HS-GC was established. The modifier can sufficiently dissolve the drugs then disperse into water. Moreover, methanol is little in the matrix medium and into the GC system and its retention time is short, which is no interference to the analytes.Although the problem of dissolution of analytes was solved by adding the modifier, there still are some shortcomings that the slight modifier—methanol volatilizes easily and can influence the headspace efficency. In the process of looking for suitable matrix media, a method for for the determination of trace menthol in moisten eye drops by HS-GC was established with 0.1% polysorbate 80 solution as matrix medium. Solubilizing agent—polysorbate 80 was added to solve the dissolubility of menthol and improve the the headspace efficiency, thus the sensitivity of the menthod. Polysorbate 80 is involatile and non-interference for the detection. 0.1% polysorbate 80 solution is expected to be an ideal matrix medium for the analysis of liposoluble drugs and volatile components by HS-GC.In recent years, ionic liquids (ILs) as a group of novel solvents, are used as ideal matrix media due to their inherent properties like negligible vapor pressure, high thermal stability and solubility, and the application of ILs as headspace matrix media for residuals analysis in pharmaceuticals are studied by our laboratory. However, because of the varieties of ILs it is a difficult issue to choose a suitable one among the enormous ILs family. As a result, the recent applications still remain at a"trying-comparing-screening"stage up to now, which will inevitably lead to the waste of time and cost. In addition, the theoretical study is relatively weak and there are still no investigations of quantitative theoretical studies on the partition coefficients for residual solvents between ILs and headspace gas phase by HS-GC. In this paper, the quantitative prediction of the partitioning behaviors of eight residual solvents (aliphatic alcohols) between three ILs and headspace gas phase by HS-GC was studied. The mathematical model of the partition coefficients (K) and parameters of physical properties of these solvents was initially established. It provided a helpful guidance for the application of ionic liquids as matrix media to the analysis of solvents by HS-GC.Through the stydy of the modifier behavior of water, the problem of dissolution of analytes was solved and the headspace efficency was still ensured. The methods for the determing the volatile components in pharmaceuticals were established. The disadvantages of these complicated extractions were avoided, but there are still some shortcomings, such as the increased sensitivity can be limited since the analytes are determined by only one distribution from headspace matrix medium. To further improve the accuracy and sensitivity, we try to use headspace single drop microextraction (HS-SDME) for the analysis of volatile components in pharmaceuticals. In this sense, analytes are determined through the twice distribution and concentration, then a higher sensitivity can be acquired. HS-SDME is a simple, rapid and sensitive sample pretreatment method for the analysis of volatile components in pharmaceuticals. Thence, after the investigation and optimization of the extraction conditions, a method for the determination of menthol and methyl salicylate in Baicao oils by HS-SDME-GC with DMF as extraction solvent. The proposed method is simple, fast, accurate and sensitive without complicated sample pretreatment, and the extraction and purification in an airtight space is achieved in 5 min.Although HS-SDME presents lots of advantages, evaporation of the extracting organic solvent drop is an important drawback that deteriorates the method figures of merit. The extraction volume is small and it is difficult to suspend under high temperature for long time. Furthermore, for full utilization the advantages of ILs such as low vapor pressure, high stability, large viscosity and density, an improved HS-SDME procedure using ILs has been developed for the pretreatment of volatile components with high boiling point in pharmaceuticals. The volume of ionic liquid microdrop was increased by modifying the device of suspended drop to increase the contact area of the microdrop and the bottom of plastic tube to the tip of microsyringe needle. The stability of microdrop and the sensitivity of the method were improved. The procedure combined with HPLC was applied to the determination of guaiacol in Compoud Succus Bambusae Oral Solution and camphor and trans-anethole in compound liquorice tablets. It is proved to be not only simply, fast, accurate, and inexpensive, but also with no complicated sample pretreatment and no contaimination.In summary, the new sample pretreatment methods in this paper for the analysis of volatile components in pharmaceuticals can make up disadvantages of other sample pretreatment methods before. Simple, rapid and sensitive methods have been provided for quality control of volatile components in pharmaceuticals.PART 1 Study and Application of Traditional Headspace Matrix Media for Volatile Components in Pharmaceuticals by HS-GC ⅠWater as Headspace Matrix Media for Determining Residual Solvent Ethanol in Huoxiang Zhengqi Oral Liquid by HS-GCObjective: To solve problems of the contaimination for analysis system and interference to the analytes, a method was estabilishde to determine residual solvent ethanol in Huoxiang Zhengqi oral liquid by HS-GC.Methods: Optimal headspace conditions were observed when the vials were equilibrated at 80℃for 20 min with water as matrix medium. The injection volume of 0.5 mL.The chromatographic analysis was carried out on a DM-WAX capillary column (30 m×0.32 mm×0.25μm) with FID detector. The column, injector and detector temperature were set at 70℃, 150℃, and 200℃, respectively.Results: The method avoids the disadvantages of direct injection. An wide linear range, good recoveries and high sensitivity were acquired. Method validation showed that the calibration curve was linear in the range of 1.95~5.00×102μg·mL-1 for ethanol (r=0.999 9). The average recovery was 96.5% with RSD of 1.5%. The detection limit was 0.122μg·mL-1.Conclusion: The proposed method is proved to be simple, accurate, and sensitive. Water as matrix medium, the wide chromatographic peak which may be produced by DMF, DMSO, etc. are avoided. It not only decreases the interference to the analytes but aslo has no contaimination for GC system, which is suitable for the determination of residual solvent ethanol in Huoxiang Zhengqi oral liquid.ⅡWater Added Modifier as Headspace Matrix Media for Determining Menthol and Methyl Salicylate in Luofushan Baicao Oils by HS-GCObjective: By adding the modifier in matrix medium to solve the bad dissolubility of drug—Baicao oils and volatile components—menthol and methyl salicylate. To establish a simple and rapid method for the determination of menthol and methyl salicylate in Luofushan Baicao oils by HS-GC.Methods: A special sample pretreatment method was performed by injecting 50μL of its methanol dilution solution with mcroliter syringes in 2 mL matrix medium—water, then to detect by headspace injection directly. The analytes were equilibrated with oven temperation 80℃for 30 min. The injection volume was 1.0 mL. The gas chromatograph was equipped with a FID detector, a 30 m×0.53 mm×0.50μm capillary column with PEG-20M as the stationary phase. The column oven temperature program involved an initial temperature of 100℃for 3 min, then increased at 4℃/min to 112℃, and at 10℃/min to 152℃, respectively, and held for 5 min. The injector and detector temperature were set at 200℃, and 230℃, respectively.Results: An excellent seperation of menthol, methyl salicylate and other contents was achieved by HS-GC without complicated extraction. The calibration curves were linear in the range of 62.4-9.98×102μg·mL-1 for menthol (r=0.999 6) and 1.25×102~2.00×103μg·mL-1 for methyl salicylate (r=0.999 0) respectively. The average recoveries were 99.2% and 98.1% for two above mentioned components respectively with RSD of 0.5% and 0.9%. The detection limits were 1.95μg·mL-1, 7.81μg·mL-1, respectively.Conclusion: The method is simple, rapid and accurate. By adding the modifier-methanol in matrix medium, the bad dissolubility of drug was solved. Moreover, methanol is little in the matrix medium and into the GC system and its retention time is short, which is no interference to the analytes. It is suitable for the quality control of Luofushan Baicao oils.ⅡWater Added Solubilizing Agent as Headspace Matrix Media for Determining of Menthol in Moisten Eye Drops by HS-GCObjective: By adding solubilizing agent-polysorbate 80 to solve the dissolubility of menthol and improve the sensitivity of the menthod. A headspace gas chromatography was established for the determination of trace menthol in moisten eye drops. Methods: A 30 m×0.53 mm×0.50μm SMA-WAX capillary column with PEG-20M as the stationary phase was used with FID detector when the vials were equilibrated at 85℃for 20 min with 0.1% polysorbate 80 solution as matrix medium, and injection volume of 1.0 mL. The column, injector and detector temperature were set at 125℃, 150℃, and 170℃, respectively.Results: A wide linear range, good recoveries and high sensitivity were acquired without complicated pretreatments. Method validation showed that the calibration curve was linear in the concentration range of 2.4~76.8μg·mL-1 (r=0.999 4),The recovery was between 100.4 and 101.1%, and RSD were less than 1.5%. The detection limit was 9.45μg·L-1.Conclusion: The proposed method is simple, fast, and accurate. 0.1% polysorbate 80 solution is an ideal matrix medium which will improve the dissolubility of menthol and the sensitivity of the menthod. The method is suitable for the quality control of trace menthol in ophthalmic solutions.PART 2 Theoretical Study of Novel Ionic Liquids as Matrix Media for The Partitioning Behavior of Volatile Components in Pharmaceuticals—Organic Residual Solvents by HS-GC: Model of Quantitative Prediction of Ionic Liquid-Gas Partition CoefficientsObjective: An experimental and modeling study was carried out for the quantitative prediction of the partitioning behaviors of some organic residual solvents between ionic liquids and gas phase by headspace gas chromatography (HS-GC) , and an ionic liquids model with ideal headspace partitioning behaviors will be established.Methods: Three ionic liquids commonly used were introduced as matrix medium for eight aliphatic alcohols. The analyte were equilibrated at the temperature of 100℃in an oven for 30 min. A 0.5 mL injection volume was used. A phenomenex ZB-1 fused silica capillary column (60 m×0.53 mm×5.00μm) was utilized for chromatographic separation of the solvents. The initial column temperature was 50℃for 2 min and then increased to 130℃at a rate of 5℃/min and held for 2 min. The injector was maintained at 180℃with a splitless injection. The FID was set at 200℃.Results: K was calculated by the experiment. The analyses were carried out with the SPSS version 13.0 program. A general model was established to predict the ionic liquid—gas partition coefficients (K) for these solvents in this paper. Parameters of physical properties of these solvents such as Tb (the temperature of boiling points) and logPO/W (the logarithm of the octanol/water partition coefficient for a solvent) were used in the model. Three regression equations were obtained and the multiple coefficients correlation were in all cases higher than 0.988Conclusion: Other ILs and solvents can be studied by the proposed method to interpret the partitioning behavior of solvents between ILs and gas phase. Since the partition coefficients have a negative effect on the sensitivity, an easier and faster method was developed by selecting ILs as matrix media for the determination of residual solvents by HS-GC.PART 3 Study and Application of the Analysis of Active Volatile Components in Pharmaceuticals by HS-SDMEⅠDMF as Extraction Solvent for Rapid Analyzing Volatile Components in Baicao Oils by HS-SDME-GCObjective: For further improving the sensitivity of the method, a simple, fast method based on headspace single drop microextraction (HS-SDME) was described here, used for the determination of volatile components such as menthol and methyl salicylate in Baicao oils by GC.Methods: A special sample pretreatment method was performed by adding 10μL of its methanol dilution solution into 5 mL water, a 1.5μL microdrop of DMF with benzyl alcohol as internal standard, exposing 5 min at extraction temperature of 40℃. After extraction, 0.5μL extractant was directely injected into GC for analysis. The determination was carried on a capillary column (30 m×0.53 mm) with PEG as the stationary phase with FID as the detector. The column oven temperature program involved an initial temperature of 100℃for 3 min, then increased at 4℃/min to 112℃, and at 10℃/min to 152℃, respectively, and held for 5 min. The injector and detector temperature were set at 200℃, and 230℃, respectively.Results: The excellent separation and detection of the target components was accomplished. The average recoveries varied between 95.4% and 99.2%, and relative standard deviations were less than 1.9%. The calibration curves were linear in the range of 1.26~80.5μg·mL-1 for menthol (r=0.9990) and 2.48~1.59×102μg·mL-1 for methyl salicylate (r=0.9991) respectively. The limits of detection for the two target analytes were found to be 7.86×10-2μg·mL-1and 0.156μg·mL-1, respectively. The sensitivity of this method is higher than direct HS-GC.Conclusion: The proposed method is simple and fast without complicated sample pretreatment, and the extraction and purification is achieved in 5 min. Since it is liquid injected an internal standard is added in the extraction solvent, it is accurate and expected to be widely applied for the analysis of volatile components in traditional chinese medicines.ⅡNovel Ionic liquid as Extraction Solvent for Determination of Volatile Components with High Boiling Point in Traditional Chinese Medicines by HS-SDME-HPLCObjective: An improved HS-SDME procedure using ionic liquid has been developed for the pretreatment of volatile components with high boiling point in traditional Chinese medicines. The procedure combined with HPLC was applied to the determination of guaiacol in Compoud Succus Bambusae Oral Solution.Methods: The volume of ionic liquid microdrop was increased to 12μL by modifying the device of suspended drop to increase the contact area of the microdrop and the bottom of plastic tube to the tip of microsyringe needle. The stability of microdrop and the sensitivity of the method were improved. A 12μL ionic liquid [BMIM][PF6] microdrop was exposed on the headspace above a 5 mL sample solution containing 36% (w/v) sodium chloride with a stirring rate of 1000 rpm at 80℃for 30 min. After the extraction, the microdrop was directly injected into HPLC for analysis. Chromatographic separation was achieved on a Kromasil C18 column (150 mm×4.6 mm I.D., 5μm particle size) using 1% acetic acid aqueous solution-methanol (70:30 v/v) as the mobile phase with a flow rate of 1.0 mL/min. A room temperate was used and the elutes were detected by UV at 274 nm.Results: In the optimum experimental conditions, a good linear relation was obtained in the range of 5.04×10-2~1.61μg·mL-1 for guaiacol (r=0.9997). The limit of detection was 0.394μg·L-1 and the average recovery was 97.6% with the relative standard deviations of 2.5%.Conclusion: The proposed method is proved to be not only simply, fast, accurate, and inexpensive, but also with no complicated sample pretreatment and no contaimination.ⅢAn Improved Ionic Iiquid-based HS-SDME-HPLC for the Analysis of Camphor and Trans-anethole in Compound Liquorice TabletsObjective: A simple, accurate, and sensitive ionic liquid-based HS-SDME procedure followed by HPLC was developed for the determination of camphor and trans-anethole in compound liquorice tablets.Methods: The volume of ionic liquid microdrop was increased to 12μL by modifying the device of suspended drop to increase the contact areas of the microdrop and the bottom of the PTFE tube to the tip of the microsyringe needle. The stability of microdrop and the sensitivity of the method were improved. Several variables involved in the IL-HS-SDME procedure (namely ionic liquids type, microdrop volume, stirring speed, ionic strength, extraction temperature, time and etc.) were investigated. After the extraction, the resulting microdrop was directly injected onto HPLC for analysis. Chromatographic separation was achieved on a Promosil C18 column (150 mm×4.6 mm I.D., 5μm particle size) using methanol-water (70:30 v/v) as the mobile phase with a flow rate of 1 mL/min. The column temperate was set at 35℃and the analytes were detected by UV at 289 nm.Results: Under the optimum experimental conditions, the calculated calibration curves gave acceptable linearity for camphor and trans-anethole with the concentration range from 0.156~10.0μg·mL?1 (r=0.9990, and 0.9998, respectively). The results showed excellent recovery ranging from 98.5 to 101.2% with RSD of less than 4.2%. The limits of detection for the two target analytes were found to be 9.77 and 1.95×10-2μg·L?1, respectively.Conclusion: In this study, the separation, purification and enrichment were achieved in one step in a airtight system, which reduced the interferences caused by other complicated constituents, increased the signal-to-noise of the method, and ensured the accuracy of the results since there was no loss of volatile components. This procedure has been successfully used for the analysis of camphor and trans-anethole in compound liquorice tablets and is expected to be widely applied for sample pretreatment of volatile components with high boiling points in samples with complicated matrix, for example, the extractions of plants, Chinese traditional drugs, etc.