Pub Date : 2017-06-12DOI: 10.4172/2155-9872.1000366
Yi Zhu, T. Feng, Y. Diao, Chunyan Tu
Linaclotide, a synthetic peptide, is a guanylate cyclase-C (GC-C) agonist for treating irritable bowel syndrome with constipation and chronic idiopathic constipation. A simple, precise and selective RP-HPLC method for linaclotide analysis was developed and validated. Analysis was achieved with a SinoChrom ODS-BP column, mobile phase A and B comprised 30 mol·L-1 phosphate (pH 2.8) solution and acetonitrile respectively, gradient elution B% set to 7%?25% from 0 to 32 min and to 25% from 32 to 50 min, flow rate of 1.0 mL·min-1, oven temperature at 40?, injection volume of 20 µL, and UV detection wavelength at 214 nm. Method validation was carried out in linearity, LOD, LOQ, precision and stability. Modified method for linaclotide preparation on a preparative liquid chromatography was developed from the analytical method and applied successfully.
利那克洛肽是一种合成肽，是一种鸟苷酸环化酶- c (GC-C)激动剂，用于治疗肠易激综合征伴便秘和慢性特发性便秘。建立了一种简便、精确、选择性好的反相高效液相色谱法分析利那洛肽。采用SinoChrom ODS-BP色谱柱进行分析，流动相a和B分别为30 mol·L-1磷酸(pH 2.8)溶液和乙腈，梯度洗脱B%设置为7%?0 ~ 32 min 25%， 32 ~ 50 min 25%，流速1.0 mL·min-1，烘箱温度40?，进样量20 µL，紫外检测波长214 nm。对方法进行了线性度、定量限、定量限、精密度和稳定性验证。在分析方法的基础上，建立了制备型液相色谱法制备利那洛肽的改进方法，并成功应用。
{"title":"Analysis and Preparation of Linaclotide by High Performance Liquid Chromatography","authors":"Yi Zhu, T. Feng, Y. Diao, Chunyan Tu","doi":"10.4172/2155-9872.1000366","DOIUrl":"https://doi.org/10.4172/2155-9872.1000366","url":null,"abstract":"Linaclotide, a synthetic peptide, is a guanylate cyclase-C (GC-C) agonist for treating irritable bowel syndrome with constipation and chronic idiopathic constipation. A simple, precise and selective RP-HPLC method for linaclotide analysis was developed and validated. Analysis was achieved with a SinoChrom ODS-BP column, mobile phase A and B comprised 30 mol·L-1 phosphate (pH 2.8) solution and acetonitrile respectively, gradient elution B% set to 7%?25% from 0 to 32 min and to 25% from 32 to 50 min, flow rate of 1.0 mL·min-1, oven temperature at 40?, injection volume of 20 µL, and UV detection wavelength at 214 nm. Method validation was carried out in linearity, LOD, LOQ, precision and stability. Modified method for linaclotide preparation on a preparative liquid chromatography was developed from the analytical method and applied successfully.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72857195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-05-24DOI: 10.4172/2155-9872.1000365
E. Abramov, Ouri Schwob, Ofra Benny
Pathological angiogenesis is a critical component in cancer, in chronic systemic inflammatory diseases such as psoriasis and rheumatoid arthritis, and in ocular diseases. Anti-angiogenic drugs have the ability to prevent, inhibit, and regress newly formed blood vessels. The activity of TNP-470 (chloro acetylcarbamoylfumagillol), a potent anti-angiogenic drug, has been demonstrated in numerous preclinical studies and in eight clinical studies involving more than three hundred patients. Despite its encouraging efficacy, TNP-470 is unstable compound with short plasma half-life, and, as was found clinically it can cause neurotoxicity side-effects at high doses. In light of these limitations, developing a transdermal drug delivery for TNP-470, can offer a novel and promising clinical usage for this drug by improving its bioavailability, controlled dosage and safety profile. In this work, we developed a reliable method for skin permeation studies of TNP-470, using the pig skin in Franz diffusion cells and High-Performance Liquid Chromatography (HPLC) analysis. Additionally, we performed a broad stability and degradation studies of TNP-470 in different mediums and identify optimal stabilizing conditions in acetate buffer pH-4.5, which can be used for transdermal formulation. Our results demonstrated excellent permeability properties of TNP-470 through the pig skin, where 25% from the initial amount was crossed through the skin membrane after 72 hours. Our results suggesting that TNP-470 is a good candidate for transdermal drug delivery, whereas, an optimal dermal formulation would improve drug’s pharmacokinetic properties and toxicity profile by introducing it in a slow release system.
{"title":"Analytical Method for Transdermal Delivery of the Anti-angiogenic CompoundTNP-470","authors":"E. Abramov, Ouri Schwob, Ofra Benny","doi":"10.4172/2155-9872.1000365","DOIUrl":"https://doi.org/10.4172/2155-9872.1000365","url":null,"abstract":"Pathological angiogenesis is a critical component in cancer, in chronic systemic inflammatory diseases such as psoriasis and rheumatoid arthritis, and in ocular diseases. Anti-angiogenic drugs have the ability to prevent, inhibit, and regress newly formed blood vessels. The activity of TNP-470 (chloro acetylcarbamoylfumagillol), a potent anti-angiogenic drug, has been demonstrated in numerous preclinical studies and in eight clinical studies involving more than three hundred patients. Despite its encouraging efficacy, TNP-470 is unstable compound with short plasma half-life, and, as was found clinically it can cause neurotoxicity side-effects at high doses. In light of these limitations, developing a transdermal drug delivery for TNP-470, can offer a novel and promising clinical usage for this drug by improving its bioavailability, controlled dosage and safety profile. In this work, we developed a reliable method for skin permeation studies of TNP-470, using the pig skin in Franz diffusion cells and High-Performance Liquid Chromatography (HPLC) analysis. Additionally, we performed a broad stability and degradation studies of TNP-470 in different mediums and identify optimal stabilizing conditions in acetate buffer pH-4.5, which can be used for transdermal formulation. Our results demonstrated excellent permeability properties of TNP-470 through the pig skin, where 25% from the initial amount was crossed through the skin membrane after 72 hours. Our results suggesting that TNP-470 is a good candidate for transdermal drug delivery, whereas, an optimal dermal formulation would improve drug’s pharmacokinetic properties and toxicity profile by introducing it in a slow release system.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81342585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-05-22DOI: 10.4172/2155-9872.1000364
Miguel Ossandon, Joshua Balsam, Hugh Alan Bruck, A. Rasooly, K. Kalpakis
Identification of Circulating Tumor Cells (CTCs) has shown promising clinical applications, but since CTCs are found in very low concentration in blood large sample volumes are needed for meaningful enumeration. This issue impedes the analysis of CTCs using standard flow cytometry due to its low throughput. To address this issue, a high throughput microfluidic cytometer was recently developed using a wide field flow- flow cell instead of the conventional narrow hydrodynamic focusing cells (used in traditional flow cytometry) enabling analysis of large volumes at lower flow rate. This wide-field flow cytometer adopts a technique known as “streak photography” where exposure times and flow velocities are set such that the particles are imaged as short “streaks”. Since streaks are imaged with large number of pixels, they are easily distinguished from the noise which appears as “speckles” increasing the detection capabilities of the device, making it more suitable for analysis using current low sensitivity, high noise webcams or mobile phone cameras. The non-stationary nature of the high noisy background found in streak cytometry introduces additional challenges for automated cell counting methods using traditional cell detection techniques such TLC, CellProfiler, CellTracker and other tools based in traditional edge detection (e.g., Canny based filters) or manual thresholding. In order to address this issue, we developed a new automated enumeration approach that does not rely on edge detection or manual thresholding of individual cells, rather is based in image quantizing, morphological operations, 2D order-statistic filtering and decisions rules that take into account knowledge of the structure and expected location of the streaks in consecutive frames. We evaluated our approach comparing it with two current methods representing the major computational modalities for cell detection: CellTrack (based in edge detection) and MTrack2 (based in manual thresholding). Samples of 1 cell/mL nominal concentration were analyzed in batch size of 30 mL at flow rate of 10 mL/min and imaged at 4 frames per second (fps), the files were saved in uncompressed AVI format files. The cells were annotated and the signal to noise ratio (SNR) was calculated. For samples with average SNR greater than 4.4 dB, our method achieved a sensitivity of 91% compared to CellTrack (60%) and MTrack2 (71%). The True Positive Rate (TPR) of cells detected was 0.93 for our method compared with 0.80 for Mtrack2 and 0.29 for CellTrack. This demonstrated the ability of the algorithm to count rare cells with high accuracy for concentrations of 1 cell/mL with SNR greater than 4.4 dB. This cell counting capability will enable to automate low cost imaging flow cytometry based on CCD detector and the expansion of cell-based clinical diagnostics in resource-poor settings.
{"title":"Evaluation of a Methodology for Automated Cell Counting for Streak ModeImaging Flow Cytometry","authors":"Miguel Ossandon, Joshua Balsam, Hugh Alan Bruck, A. Rasooly, K. Kalpakis","doi":"10.4172/2155-9872.1000364","DOIUrl":"https://doi.org/10.4172/2155-9872.1000364","url":null,"abstract":"Identification of Circulating Tumor Cells (CTCs) has shown promising clinical applications, but since CTCs are found in very low concentration in blood large sample volumes are needed for meaningful enumeration. This issue impedes the analysis of CTCs using standard flow cytometry due to its low throughput. To address this issue, a high throughput microfluidic cytometer was recently developed using a wide field flow- flow cell instead of the conventional narrow hydrodynamic focusing cells (used in traditional flow cytometry) enabling analysis of large volumes at lower flow rate. This wide-field flow cytometer adopts a technique known as “streak photography” where exposure times and flow velocities are set such that the particles are imaged as short “streaks”. Since streaks are imaged with large number of pixels, they are easily distinguished from the noise which appears as “speckles” increasing the detection capabilities of the device, making it more suitable for analysis using current low sensitivity, high noise webcams or mobile phone cameras. The non-stationary nature of the high noisy background found in streak cytometry introduces additional challenges for automated cell counting methods using traditional cell detection techniques such TLC, CellProfiler, CellTracker and other tools based in traditional edge detection (e.g., Canny based filters) or manual thresholding. In order to address this issue, we developed a new automated enumeration approach that does not rely on edge detection or manual thresholding of individual cells, rather is based in image quantizing, morphological operations, 2D order-statistic filtering and decisions rules that take into account knowledge of the structure and expected location of the streaks in consecutive frames. We evaluated our approach comparing it with two current methods representing the major computational modalities for cell detection: CellTrack (based in edge detection) and MTrack2 (based in manual thresholding). Samples of 1 cell/mL nominal concentration were analyzed in batch size of 30 mL at flow rate of 10 mL/min and imaged at 4 frames per second (fps), the files were saved in uncompressed AVI format files. The cells were annotated and the signal to noise ratio (SNR) was calculated. For samples with average SNR greater than 4.4 dB, our method achieved a sensitivity of 91% compared to CellTrack (60%) and MTrack2 (71%). The True Positive Rate (TPR) of cells detected was 0.93 for our method compared with 0.80 for Mtrack2 and 0.29 for CellTrack. This demonstrated the ability of the algorithm to count rare cells with high accuracy for concentrations of 1 cell/mL with SNR greater than 4.4 dB. This cell counting capability will enable to automate low cost imaging flow cytometry based on CCD detector and the expansion of cell-based clinical diagnostics in resource-poor settings.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73932274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-05-15DOI: 10.4172/2155-9872.1000363
P. K. Sahu
A chromatographic method newly optimized to identify and assay four antihypertensive drugs in tablet dosage forms was complemented by a robustness test. The best system suitability criteria for numerous responses were evaluated on the basis of the robustness test results. Generally speaking, it is difficult to achieve a total satisfactory solution. Situations may also become ambiguous if the system suitability limits for few responses of a robust method are violated. In this context, it becomes crucial to redefine these limits based on the robustness test results. In the present study, the extreme experimental (worst-case) conditions that offer worst result but still acceptable and likely to occur were predicted from the robustness test effects. Eventually, replicated experiments were executed in such worst conditions and the system suitability test (SST) limits were determined.
{"title":"Definition of System Suitability Test Limits on the Basis of Robustness TestResults","authors":"P. K. Sahu","doi":"10.4172/2155-9872.1000363","DOIUrl":"https://doi.org/10.4172/2155-9872.1000363","url":null,"abstract":"A chromatographic method newly optimized to identify and assay four antihypertensive drugs in tablet dosage forms was complemented by a robustness test. The best system suitability criteria for numerous responses were evaluated on the basis of the robustness test results. Generally speaking, it is difficult to achieve a total satisfactory solution. Situations may also become ambiguous if the system suitability limits for few responses of a robust method are violated. In this context, it becomes crucial to redefine these limits based on the robustness test results. In the present study, the extreme experimental (worst-case) conditions that offer worst result but still acceptable and likely to occur were predicted from the robustness test effects. Eventually, replicated experiments were executed in such worst conditions and the system suitability test (SST) limits were determined.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76050368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-05-12DOI: 10.4172/2155-9872.1000362
Subodh Kumar, Priyanka Mittal
Biomarker discovery is relying on the sensitivity and specificity of the detected biomolecules in clinical samples. Several potential biomarkers against the particular disease cannot be detected because of the unavailability of either specific procedure or sensitive instrument or both, which hamper the diagnosis. In spite of that some of the potential biomarkers are available in trace amount in biological fluids like serum, urine, buccal swab, sputum etc. and need a sensitive method to detect precisely those biomolecules. Now days Selected Reaction Monitoring (SRM) is Mass Spectrometry based approached who can overcome the problems associated with biomarker discovery. India have adequate and variety of clinical samples but some time due to lack of infrastructure and knowledge we are unable to utilise those samples for human welfare. In this review, we are discussing about the experimental setup and procedure of SRM experiment.
{"title":"Selected Reaction Monitoring: A Valid Tool for Targeted Quantitation of ProteinBiomarker Discovery","authors":"Subodh Kumar, Priyanka Mittal","doi":"10.4172/2155-9872.1000362","DOIUrl":"https://doi.org/10.4172/2155-9872.1000362","url":null,"abstract":"Biomarker discovery is relying on the sensitivity and specificity of the detected biomolecules in clinical samples. Several potential biomarkers against the particular disease cannot be detected because of the unavailability of either specific procedure or sensitive instrument or both, which hamper the diagnosis. In spite of that some of the potential biomarkers are available in trace amount in biological fluids like serum, urine, buccal swab, sputum etc. and need a sensitive method to detect precisely those biomolecules. Now days Selected Reaction Monitoring (SRM) is Mass Spectrometry based approached who can overcome the problems associated with biomarker discovery. India have adequate and variety of clinical samples but some time due to lack of infrastructure and knowledge we are unable to utilise those samples for human welfare. In this review, we are discussing about the experimental setup and procedure of SRM experiment.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89683661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-04-20DOI: 10.4172/2155-9872.1000E127
Q. Hu
{"title":"Editorial - Sensitivity of Analytical and Bioanalytical Techniques","authors":"Q. Hu","doi":"10.4172/2155-9872.1000E127","DOIUrl":"https://doi.org/10.4172/2155-9872.1000E127","url":null,"abstract":"","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88034954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-04-10DOI: 10.4172/2155-9872.1000360
M. Doroodmand, F. Ghasemi
A new method has been introduced based on aerosol hygroscopic growth as a new factor for trace and ultra-trace determination of phosphorous in flame containing optical trapping-cavity ring down aerosol extinction (emission) spectrometer (OT-CRD-AES). In this study, a cavity ring down has been designed using hydrogen and air as fuel and oxidant during introduction of the aerosols containing phosphorous species using an ultrasonic generator (humidifier) from an acidic solution by a flow rate of N2 , followed by detection of the Mie scattering using a charged coupling device (CCD) system. Parameters having strong influence during following scattering of the aerosols during their hygroscopic growth inside the humidified flame (H2 /air), include: influence and amount of Na+ as radiation buffer (as light source), flow rates of H2 , air and N2 , kind and concentrations of acid, evaluation of the aerosols inside flame, etc. These parameters were optimized using simplex and one at a time methods. Based on the figures of merit under optimized condition, two linear calibration curves with reverse slopes were evaluated between 10.0 - 250.0 ng mL-1 and 1.0 - 20.0 µg mL-1 with correlation coefficients (R2 ) the same as 0.999 and 0.998, respectively. The calibration sensitivities were also estimated to 57.46 and 0.348 (a.u), respectively, with detection limit of 5.0 ng mL-1. The mechanism of the radiation (Mie scattering) was also evidenced based on i) dependency of the scattered radiations to the quantity of an alkali ions such as Na+ as well as the humidity, ii) presence of acceptable correlation between the response of the cavity with turbidometry, iii) observation of blue shift from green (color related to the luminescence of HPO* in H2 /air flame) to blue (scattered radiation) and finally iv) effect of hydration number during stability and growth of the aerosols inside the flames. No serious interference was evaluated during analysis of at least 500-fold excess of various foreign species. However, the only observed interference was evaluated during introduction of 200-fold excess of SO4 2-. Good correlation was also evaluated between the results obtained from this technique and ion exchange chromatography during analysis of wastewater samples that clearly revealed the reliability of this method for phosphorous detection and determination at µg mL-1 and ng mL-1 levels.
介绍了一种以气溶胶吸湿生长为新因子的火焰中痕量和超痕量磷的测定方法-光学捕获腔环形气溶胶消光(发射)光谱仪。在本研究中，利用超声波发生器(加湿器)以氮气的流速从酸性溶液中引入含磷气溶胶，并利用带电耦合装置(CCD)系统检测Mie散射，以氢气和空气作为燃料和氧化剂设计了一个腔环。对气溶胶在加湿火焰(H2 /空气)内吸湿生长过程中后续散射影响较大的参数包括:作为辐射缓冲剂(作为光源)的Na+的影响和数量、H2、空气和N2的流速、酸的种类和浓度、火焰内气溶胶的评价等。采用单纯形法和一次一个法对这些参数进行了优化。以优化条件下的优值图为基础，在10.0 ~ 250.0 ng mL-1和1.0 ~ 20.0µg mL-1范围内建立了具有反斜率的线性校准曲线，相关系数(R2)分别为0.999和0.998。校准灵敏度分别为57.46和0.348 (a.u)，检出限为5.0 ng mL-1。辐射(Mie散射)的机制也得到了证明，基于i)散射辐射与碱离子(如Na+)的数量以及湿度的依赖关系，ii)在腔的响应与浊度测量之间存在可接受的相关性。iii)观察蓝色从绿色(与H2 /空气火焰中HPO*的发光有关的颜色)到蓝色(散射辐射)的转变，最后iv)火焰内气溶胶在稳定和生长过程中水化数的影响。在分析各种外来种至少超过500倍时，没有评估严重的干扰。然而，唯一观察到的干扰是在引入超过200倍的so42 -时评估的。在废水样品分析中，该技术与离子交换色谱法的结果之间也有良好的相关性，这清楚地表明该方法在µg mL-1和ng mL-1水平下的磷检测和测定是可靠的。
{"title":"Aerosol Hygroscopic Growth as a New Factor for Trace and Ultra-TraceDetermination of Phosphorous in Flame Containing Optical Trapping-CavityRing-Down Spectroscopy","authors":"M. Doroodmand, F. Ghasemi","doi":"10.4172/2155-9872.1000360","DOIUrl":"https://doi.org/10.4172/2155-9872.1000360","url":null,"abstract":"A new method has been introduced based on aerosol hygroscopic growth as a new factor for trace and ultra-trace determination of phosphorous in flame containing optical trapping-cavity ring down aerosol extinction (emission) spectrometer (OT-CRD-AES). In this study, a cavity ring down has been designed using hydrogen and air as fuel and oxidant during introduction of the aerosols containing phosphorous species using an ultrasonic generator (humidifier) from an acidic solution by a flow rate of N2 , followed by detection of the Mie scattering using a charged coupling device (CCD) system. Parameters having strong influence during following scattering of the aerosols during their hygroscopic growth inside the humidified flame (H2 /air), include: influence and amount of Na+ as radiation buffer (as light source), flow rates of H2 , air and N2 , kind and concentrations of acid, evaluation of the aerosols inside flame, etc. These parameters were optimized using simplex and one at a time methods. Based on the figures of merit under optimized condition, two linear calibration curves with reverse slopes were evaluated between 10.0 - 250.0 ng mL-1 and 1.0 - 20.0 µg mL-1 with correlation coefficients (R2 ) the same as 0.999 and 0.998, respectively. The calibration sensitivities were also estimated to 57.46 and 0.348 (a.u), respectively, with detection limit of 5.0 ng mL-1. The mechanism of the radiation (Mie scattering) was also evidenced based on i) dependency of the scattered radiations to the quantity of an alkali ions such as Na+ as well as the humidity, ii) presence of acceptable correlation between the response of the cavity with turbidometry, iii) observation of blue shift from green (color related to the luminescence of HPO* in H2 /air flame) to blue (scattered radiation) and finally iv) effect of hydration number during stability and growth of the aerosols inside the flames. No serious interference was evaluated during analysis of at least 500-fold excess of various foreign species. However, the only observed interference was evaluated during introduction of 200-fold excess of SO4 2-. Good correlation was also evaluated between the results obtained from this technique and ion exchange chromatography during analysis of wastewater samples that clearly revealed the reliability of this method for phosphorous detection and determination at µg mL-1 and ng mL-1 levels.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83259256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-04-08DOI: 10.4172/2155-9872.1000358
M. EmanAbdelsayed, A. SaharMaksoud, I. Sidhom, Z. MohamedGad, S. RashaHanafi
The need of a robust, sensitive HPLC method for the quantitation of 6-thioguaninenucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) is indispensable to relate levels of these metabolites with emergence of signs of toxicity in patients undergoing treatment with 6-mercaptopurine (6-MP), paving the road to accurate dose calculations and thus providing a cost-effective treatment approach. Previously reported methods were either laborious, required special types of C18 columns, or had long run times. A Design of Experiments (DoE) approach targeting the shortest run time with greatest selectivity was adopted using a user friendly HPLC method development simulation software (DryLab®). Analytes eluted within 10 min, at 3.8, 4.2, 5.6 and 7.5 min for 6-TG, 6-MP, 6-MMP and Dithiothreitol (DTT) respectively. Excellent recovery percentages of 90.9 ± 14.4, 87.8 ± 6.7 and 92.1 ± 9.08, respectively were obtained. The method proved its validity and robustness according to the International Conference on Harmonization (ICH) guidelines. The LOD of 6-MP, 6-TG and 6-MMP were 6, 9 and 24 pmol/8 × 108 RBCs, respectively. Twenty-Two Acute Lymphocytic Leukemia (ALL) children recruited from 57357 Cancer Hospital (Cairo, Egypt) had their 6-MP metabolites measured using the developed method. A strong negative correlation was manifested between TG and RBCs count and hemoglobin (p=0.009 and 0.002 respectively). WBC and neutrophils showed a negative correlation to TG at Continuation 1 phase of treatment, confirming the association of TG with myelotoxicity. The significant correlation between MMP and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (p=0.030, 0.004) explained its potential hepatotoxicity.
{"title":"HPLC Determination of the Levels of 6-Mercaptopurine Metabolites Suitablefor the Clinical Risk Assessment of its Toxicity among Egyptian Children withAcute Lymphocytic Leukemia","authors":"M. EmanAbdelsayed, A. SaharMaksoud, I. Sidhom, Z. MohamedGad, S. RashaHanafi","doi":"10.4172/2155-9872.1000358","DOIUrl":"https://doi.org/10.4172/2155-9872.1000358","url":null,"abstract":"The need of a robust, sensitive HPLC method for the quantitation of 6-thioguaninenucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) is indispensable to relate levels of these metabolites with emergence of signs of toxicity in patients undergoing treatment with 6-mercaptopurine (6-MP), paving the road to accurate dose calculations and thus providing a cost-effective treatment approach. Previously reported methods were either laborious, required special types of C18 columns, or had long run times. A Design of Experiments (DoE) approach targeting the shortest run time with greatest selectivity was adopted using a user friendly HPLC method development simulation software (DryLab®). Analytes eluted within 10 min, at 3.8, 4.2, 5.6 and 7.5 min for 6-TG, 6-MP, 6-MMP and Dithiothreitol (DTT) respectively. Excellent recovery percentages of 90.9 ± 14.4, 87.8 ± 6.7 and 92.1 ± 9.08, respectively were obtained. The method proved its validity and robustness according to the International Conference on Harmonization (ICH) guidelines. The LOD of 6-MP, 6-TG and 6-MMP were 6, 9 and 24 pmol/8 × 108 RBCs, respectively. Twenty-Two Acute Lymphocytic Leukemia (ALL) children recruited from 57357 Cancer Hospital (Cairo, Egypt) had their 6-MP metabolites measured using the developed method. A strong negative correlation was manifested between TG and RBCs count and hemoglobin (p=0.009 and 0.002 respectively). WBC and neutrophils showed a negative correlation to TG at Continuation 1 phase of treatment, confirming the association of TG with myelotoxicity. The significant correlation between MMP and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (p=0.030, 0.004) explained its potential hepatotoxicity.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84301909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-04-08DOI: 10.4172/2155-9872.1000359
Yan Wang, Peng Feng, Z. Sosic, Li Zang
Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, we describe a fast and simple microfluidic ZipChip CE-MS method to measure quality attributes of monoclonal antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized with charge-based separation using microfluidic capillary electrophoresis coupled to a high-resolution mass spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using the same method with a single microfluidic device. A total volume of ten to fifty microliter of cell culture supernatant is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLCMS analysis following Protein A (ProA) purification step has been demonstrated.
{"title":"Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples UsingZipChip CE-MS","authors":"Yan Wang, Peng Feng, Z. Sosic, Li Zang","doi":"10.4172/2155-9872.1000359","DOIUrl":"https://doi.org/10.4172/2155-9872.1000359","url":null,"abstract":"Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, we describe a fast and simple microfluidic ZipChip CE-MS method to measure quality attributes of monoclonal antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized with charge-based separation using microfluidic capillary electrophoresis coupled to a high-resolution mass spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using the same method with a single microfluidic device. A total volume of ten to fifty microliter of cell culture supernatant is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLCMS analysis following Protein A (ProA) purification step has been demonstrated.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82463463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2017-04-05DOI: 10.4172/2155-9872.1000357
Z. Xin, Binbin Du, Yanming Wang, Shengxu Qian, Weijia Li, Yuan Gao, Miao Sun, Shifang Luan, Jinghua Yin
Sugar-based amphipathic compounds (BA-CnAG) were successfully prepared. Polyurethane (PU) was grafted with glycidyl methacrylate (GMA) by the means of UV irradiation, and further modified with the BA-CnAG based on the ring opening of the epoxy groups. The surface graft polymerization was confirmed by ATR-FTIR and XPS. Water contact angle, protein adsorption, and platelet adhesion measurements were used to evaluate the hydrophilicity and hemocompatibility of the films. The results demonstrated that amphiphilic surfactant-containing polymer surfaces presented protein-resistant behavior and anti-platelet adhesion after functionalization with BA-CnAG. Besides, the hemocompatibility of the modified surface deteriorated as the length of hydrophobic chain of BA-CnAG increased.
{"title":"Hemocompatibility Evaluation of Polyurethane Film with Surface-Grafted Sugar-Based Amphipathic Compounds","authors":"Z. Xin, Binbin Du, Yanming Wang, Shengxu Qian, Weijia Li, Yuan Gao, Miao Sun, Shifang Luan, Jinghua Yin","doi":"10.4172/2155-9872.1000357","DOIUrl":"https://doi.org/10.4172/2155-9872.1000357","url":null,"abstract":"Sugar-based amphipathic compounds (BA-CnAG) were successfully prepared. Polyurethane (PU) was grafted with glycidyl methacrylate (GMA) by the means of UV irradiation, and further modified with the BA-CnAG based on the ring opening of the epoxy groups. The surface graft polymerization was confirmed by ATR-FTIR and XPS. Water contact angle, protein adsorption, and platelet adhesion measurements were used to evaluate the hydrophilicity and hemocompatibility of the films. The results demonstrated that amphiphilic surfactant-containing polymer surfaces presented protein-resistant behavior and anti-platelet adhesion after functionalization with BA-CnAG. Besides, the hemocompatibility of the modified surface deteriorated as the length of hydrophobic chain of BA-CnAG increased.","PeriodicalId":14865,"journal":{"name":"Journal of analytical and bioanalytical techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88835481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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