Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Simultaneous analysis of free phytosterols and phytosterol glycosides in rice bran by SPE/GC-MS.

In this study, free phytosterols and phytosterol glycosides in rice bran were successfully separated and analyzed using solid-phase extraction (SPE) combined with gas chromatography-mass spectrometry (GC-MS). The results showed that free phytosterols in rice bran included ergosterol (129 ± 8 µg/g rice bran), campesterol (126 ± 9 µg/g rice bran), stigmasterol (106 ± 9 µg/g rice bran), ß-sitosterol (305 ± 10 µg/g rice bran), cycloartenol (80.5 ± 3.9 µg/g rice bran) and 24-methylenecycloartenol (87.1 ± 2.2 µg/g rice bran), while phytosterol glycosides included campesterol glucoside (16.0 ± 1.3 µg/g rice bran), stigmasterol glucoside (99.0 ± 4.9 µg/g rice bran) and ß-sitosterol glucoside (133 ± 7 µg/g rice bran). The methodological validation indicated that this method could accurately quantify free phytosterols and phytosterol glycosides in rice bran. This study provided a new direction to establish a rapid and simple method for the simultaneous determination of different forms of phytosterols in foods.

Cuantificacion de arsenico por inyeccion en flujo-generacion de hidruros-espectrometria de absorcion atomica (IF-GH-EAA) previa derivatizacion con L-cisteina. Validacion y comparaci¨®o intermetodologica utilizando dos tecnicas de referencia / Arsenic quantitation by flow injection-hydride generation- atomic absorption spectrometry (FI-HG-AAS) after L-cysteine derivatization. Validation and inter-methodological comparison using two reference techniques

La presencia de Arsenico (As) en aguas de consumo representa una problematica para la salud publica en muchas regiones del mundo, incluida la Argentina. La cuantificacion de arsenico en agua de bebida y en orina se utiliza para evaluar la exposicion a este contaminante. El presente trabajo tuvo como objetivo la validacion metodologica de una tecnica para la cuantificacion de especies del As [AsV + AsIII + acido monometilarsonico (MMA) + acido dimetilarsinico (DMA)] por inyeccion en flujo-generacion de hidruros-espectrometria de absorcion atomica (IF-GH-EAA), previa derivatizacion con L-cisteina. Los resultados fueron comparados con los obtenidos utilizando dos metodologias de referencia, generacion de hidruros-espectrometria de absorcion atomica (GH-EAA) para muestras de aguas y orina, y cromatografia de alta resolucion ¨Cgeneracion de hidruros-espectrometr¨ªa de absorcion atomica (HPLC-GH-EAA) para muestras de orina. Ademas, se evaluo la selectividad de la cuantificacion por IF-GH-EAA, en presencia de otras especies quimicas del As, provenientes del consumo de alimentos producto de la pesca, a traves de un ensayo biologico. Los niveles de As hallados en las muestras de agua y de orina utilizando las tecnicas de referencia presentaron un rango de 6 a 176 ¦Ìg/L y de 143 a 3312 ¦Ìg/g de creatinina, respectivamente. Los coeficientes de Pearson resultantes de la comparacion de los datos obtenidos por IF-GH-EAA, con los logrados por los metodos de referencia fueron r = 0,9976 y r = 0,9422, para agua y orina, respectivamente. Los resultados de la prueba biologica indicaron un mayor nivel de As, debido al consumo de alimentos producto de la pesca, cuando las muestras de orina fueron previamente mineralizadas (GH-EAA), con la consecuente sobreestimacion del contenido de As proveniente del consumo de As inorganico. Este aumento no se observo cuando estas fueron analizadas por IF-GH-EAA ...

The presence of arsenic (As) in drinking water is a public health concern in many regions of the world, including Argentina. Quantification of arsenic in drinking water and urine are used to assess exposure to this pollutant. This study aimed to validate a methodology for the quantification of As species [AsV + AsIII + acid monometilarsonico (MMA) + dimetilarsinico acid (DMA)] by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS), after derivatization with L-cysteine. The results were compared with those obtained using two methods of reference, hydride generation-atomic absorption spectrometry (HG-AAS) for water and urine samples, and high performance liquid chromatography-hydride generation-atomic absorption spectrometry (HPLC-HG-AAS) for urine samples. In addition, the selectivity of quantification by FI-HG-AAS in the presence of other chemical species of As, from fishery products intake, was evaluated through a biological assay.The As level found in water and urine samples, using the techniques of reference, showed a range from 6 to 176 ¦Ìg/L and from 143 to 3312 ¦Ìg/g creatinine, respectively. Pearson coefficients resulting from the comparison of data obtained by FI-HG-AAS with those achieved by the reference methods were r = 0.9976 and r = 0.9422 for water and urine, respectively. The results of the biological test showed a higher level of As, due to consumption of food fishery product, when urine samples were previously mineralized (HG-AAS), with consequent overestimation of the inorganic arsenic consumption. When these samples were analyzed by FI-HG-AAS this fact was not observed, and the values were comparable to baseline level prior to consumption ...

Validation on Method of Microbial Limit Test of Povidone Iodine Solution / 中国药房

OBJECTIVE:To establish a microbial limit test method for povidone iodine solution.METHODS:Validation on methodology of the microbial limit test of povidone iodine solution was performed using plating method(Ⅰ),culture agent dilution method(Ⅱ),membrane-filter procedure method(Ⅲ)and sodium thiosulfate neutralization in combination with membrane-filter procedure method(Ⅳ).RESULTS:The recovery rates of all of the test organisms in method Ⅰ and method Ⅱ approached zero,less than 70% in method Ⅲ but above 85% in method Ⅳ.The growth of the control bacteria was abnormal in method Ⅱand method Ⅲ,but normal in method Ⅳ.CONCLUSION:It is advisable to adopt the method Ⅳ for microbial limit test of povidone iodine solution.

Methodological Validation on Microbil Limit Test of Dental Ulcer Pellicles of Sunset Abelmoschus Flower / 中国药房

OBJECTIVE:To validate the test condition and test method of the microbil limit test of the Dental ulcer pellicles of Sunset Abelmoschus Flower.METHODS:The methodological validation was carried out in accordance with the standard for microbil limit test stated in the appendix of China Pharmacopoeia(2005 Edition,VolumeⅠ).RESULTS:By membrane-filter procedure,the recovery rates of different test strains in the samples were all over 70%.CONCLUSION:The membrane-filter procedure can more effectively eliminate the bacteriostatic action of Dental ulcer pellicles of Sunset Abelmoschus Flower so as to result in accurate microbil limit test results.