Robustness Study and Superior Method Development and Validation for Analytical Assay Method of Atropine Sulfate in Pharmaceutical Ophthalmic Solution

Background: The robustness is a measurement of an analytical chemical method and its ability to contain unaffected by little with deliberate variation of analytical chemical method parameters. The analytical chemical method variation parameters are based on pH variability of buffer solution of mobile phase, organic ratio composition changes, stationary phase (column) manufacture, brand name and lot number variation;flow rate variation and temperature variation of chromatographic system. The analytical chemical method for assay of Atropine Sulfate conducted for robustness evaluation. The typical variation considered for mobile phase organic ratio change, change of pH, change of temperature, change of flow rate, change of column etc. Purpose: The aim of this study is to develop a cost effective, short run time and robust analytical chemical method for the assay quantification of Atropine in Pharmaceutical Ophthalmic Solution. This will help to make analytical decisions quickly for research and development scientists as well as will help with quality control product release for patient consumption. This analytical method will help to meet the market demand through quick quality control test of Atropine Ophthalmic Solution and it is very easy for maintaining (GDP) good documentation practices within the shortest period of time. Method: HPLC method has been selected for developing superior method to Compendial method. Both the compendial HPLC method and developed HPLC method was run into the same HPLC system to prove the superiority of developed method. Sensitivity, precision, reproducibility, accuracy parameters were considered for superiority of method. Mobile phase ratio change, pH of buffer solution, change of stationary phase temperature, change of flow rate and change of column were taken into consideration for robustness study of the developed method. Results: The limit of quantitation (LOQ) of developed method was much low than the compendial method. The % RSD for the six sample assay of developed method was 0.4% where the % RSD of the compendial method was 1.2%. The reproducibility between two analysts was 100.4% for developed method on the contrary the compendial method was 98.4%.

饲料检测方法中检出限、定量限的研究与应用

对检出限和定量限在饲料检测领域中应用进行了阐述,检出限和定量限在饲料行业的各类检测标准中的表述模式并未完全统一,部分标准中规定了检出限和定量限的限定条件,不同的取样质量、定容体积与稀释倍数等前处理方法会影响方法检出限与定量限的大小,部分标准只规定了最低检测浓度(如ng/mL).在使用方法标准时,通过判断方法中检出限和定量限的表述模式,正确理解其含义,才能有效地选择合适的检测方法.文中还分析了仪器检出限与方法检出限的区别,以及如何确定两项指标,利用其来评判仪器或方法的灵敏度,从而为检测人员完成分析工作提供参考.

定量检测系统临床可报告范围评价方法的探讨

目的 探讨LR,LOQ,AMR及CRR的评价方法及其意义.方法 参考EP6-A文件评价ALT检测系统的线性范围（LR）,参考EP-17A文件评价ALT检测系统的临床可报告范围（CRR）和定量检测限（LOQ）,确定评价该检测系统的LOQ,LR的上限值（HLR）和下限值（LLR）、检测HLR的最大稀释倍数,最后确定AMR和CRR.结果 通过评价得出ALT检测系统的LOQ为5 μ/L,LR的HLR和LLR分别为10.5μ/L和1 160 μ/L,对HLR处样品的最大稀释倍数为1∶5（6倍）,AMR为5～1 160 μ/L,CRR为5～6 960 μ/L.结论 对定量检测系统进行LR,LOQ,CRR及AMR的评价,可使我们清楚了解检验结果的准确度,对超过AMR上限值的样品,应采取正确的稀释比例,才能使测定的结果准确,更重要的是工作者可以做到准确、科学、合理地向临床报告检验结果,提高工作者对定量检验结果含义的认识.

液相色谱法同时测定豇豆中吡虫啉和啶虫脒残留

经过紫外扫描确定吡虫啉和啶虫脒的最大紫外吸收波长分别为270和246 nm，通过改变其比例优化出峰时间和分离度，确定V（乙腈）∶V（水）＝3∶7为流动相，建立了两种农药的标准曲线，结果表明在0.05～5μg· mL^-1范围内线性关系均良好。进行样品加标回收实验，用乙腈提取，C18固相萃取柱净化，上述条件进行下分析，0.1，0.5，1.5 mg·kg^-1三个加标浓度回收率均在75％～110％，变异系数＜5％，准确性和稳定性都较好，方法最低定量限0.01μg·g^-1，完全可以用于实际样品的检测。

基于EP-17A2的胶体金法检测粪便隐血的空白限、检出限及定量限的建立及评价

目的：参考美国临床实验室标准化委员会（CLSI）发布的《临床实验室检验程序检测能力评价指南（第二版）》（EP-17A2）文件，探讨粪便隐血（FOB）胶体金法检测能力，建立该实验室的空白限（LOB）、检出限（LOD）及定量限（LOQ），减少弱阳性标本漏检率，并为胶体金法的检测限提供定量评价方法。方法使用人游离血红蛋白（f-Hb）ELISA试剂盒检测新鲜全血制备的系列低浓度的血红蛋白溶液，制定校准曲线，将粪便隐血的空白样本及一系列的血红蛋白低浓度样本使用胶体金试纸条进行检测，利用 Nato Checker710对显色带进行定量检测，并对定量结果进行统计分析，建立胶体金法检测粪便隐血的空白限、检出限及定量限。结果空白限为99.01 ng／ml，检出限为340.48 ng／ml，定量限为354.9 ng／ml。结论参考CLSI EP17-A2文件建立的检测限比常规方法建立的检测限科学合理，优于肉眼判断结果，更能满足临床实验室的质量要求和临床早期诊疗的需求；为提高实验室质量管理水平，每批次粪便隐血应根据此方法建立检测限，以及应用于其它项目胶体金法检出限的建立。

转基因植物Real-time PCR方法检出限和定量限的研究

目前Real-timePCR(Rt-PCR)方法在转基因植物定量检测中应用最为广泛。有关该方法的检出限和定量限的计算并没有达成统一。本文采用统计模型对三种转基因植物进行实时荧光定量PCR方法检出限和定量限的研究。实验结果表明:转基因玉米NK603检出限5拷贝,定量限14拷贝;转基因棉花Mon15985检出限5拷贝,定量限12拷贝;转基因油菜Oxy235检出限4拷贝,定量限9拷贝。是利用统计学方法对转基因植物荧光定量PCR方法检出限和定量限研究的一个积极尝试,可用于其他转基因植物检测中检出限和定量限的确定。

UPLC法测定复配食品添加剂丙酸钙含量

为避免复配添加剂对滴定结果的干扰，采用高效液相色谱法测定丙酸钙的含量。试验结果表明：丙酸的质量浓度在0.05~1.00 g/L 的范围内与其峰面积呈线性关系，定量限为0.05 g/kg，回收率为97.3%~103.4%，RSD≤2.9%（n=6）。该方法精确度好，灵敏度高，样品预处理简单，是复配食品添加剂中丙酸钙含量测定的理想方法。

Method Development and Validation for the Quantification of Acrylamide in Potato Chips and Other Locally Available Food by LC-MS/MS in Bangladesh

A sensitive, precise, accurate and simple in house validated Liquid Chromatography and Tandem mass spectrometric (LC-MS/MS) analytical method was developed for the quantification of acrylamide in potato chips, a probable carcinogenic and neurotoxic compound in fried food items. Acrylamide was extracted and cleaned up by QuEChERS method, then analyzed by triple quadrupole mass spectrophotometer using positive electrospray-ionization mode and d3-Acrylamide internal standard. The analyte and internal standard were separated on a reversed-phase C18 column (100 × 2.0 mm, 2.5 μm) by gradient elution with water and methanol containing 0.1% formic acid as mobile phase. The analytical method was fully validated by assessment of linearity, specificity, precision, accuracy, selectivity and robustness with relative standard deviation of less than 4%. The calibration curves were found linear with correlation coefficient (r2) of 0.9997 over the concentration range 5 - 500 μg/L. The recoveries of Acrylamide in the concentration of 250, 400, and 500 μg/L were 97% to 104%. Based on the signal to noise ratio, the lower limit of detection (LOD) and lower limit of quantification (LOQ) were achieved 2.0 μg/L and 4.0 μg/L respectively. The proposed method was also applied to other available most popular 24 types of food (265 number of sample) first time in Bangladesh. The highest level of acrylamide has been found in fried products with the range of 197.04 μg/L to 114.63 μg/L, protein-rich food lies between 79.76 to 89.14 μg/L whereas baked food products exhibited less content in the range of 35.23 to 51.17 μg/L.

Development and Validation of a Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in a Formulation by RP-HPLC

Present study was aimed to develop and validate a reverse-phase high-performance liquid chromatography method for simultaneous determination of sitagliptin phosphate and metformin hy-drochloride in a marketed formulation. The drug separation was performed on Hibar-240, Li-chrosphere-100 C18 ODS (250 × 4.6 mm, 5 μm) column, at a flow rate of 1 mL/min. The mobile phase used was a mixture of methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30 v/v. The detection was carried out at a wavelength of 266 nm. The retention times of sitagliptin phosphate and metformin hydrochloride were found as 6.1 and 4.9 min respectively. Linear calibration curves with good correlation coefficients were obtained over the concentration ranges of 10 - 50 μg/mL for sitagliptin and 20 - 100 μg/mL for metformin. The limit of detection was 0.016 and 0.14 μg/mL and the limit of quantification was 0.048 and 0.42 μg/mL for sitagliptin phosphate and metformin hydrochloride respectively. Validation of the method demonstrated system selectivity, specificity, linearity, accuracy and precision. The developed method was found useful in the simultaneous analysis of sitagliptin phosphate and metformin hydrochloride in formulation.

非原厂试剂在西门子ADVIA2400预稀释模式下的检测限分析

目的:对非原厂试剂在西门子ADVIA2400全自动生化分析仪的预稀释模式下的检测限进行分析.方法:以葡萄糖(Glu)和钙(Ca)为例,参考CLSI EP17-A文件,将空白样品及葡萄糖(Glu)和钙(Ca)的系列低浓度样品在西门子ADVIA2400全自动生化分析仪的预稀释模式下进行测定,对试验所得的数据采用相应的统计学方法,计算出Glu和Ca的空白限(LoB)、检出限(LoD)和定量检出限(LoQ).结果:根据EP17-A的方案,统计得出Glu的LoB为0.004mmol/L,LoD为0.017mmol/L,LoQ为0.177mmol/L;Ca的LoB为0.012mmol/L,LoD为0.030mmol/L,LoQ为0.500mmol/L.结论:根据EP17-A文件中的方法,测定出Glu和Ca的实际LoQ均高于试剂说明书声明的检测下限.

Development and Validation of Amlodipine Impurities in Amlodipine Tablets Using Design Space Computer Modeling

A rapid, sensitive, robust, rugged and linear HPLC method is developed using QbD approach and validated as per ICH for the estimation of amlodipine impurities in tablet dosage form. Phosphate buffer with triethyl amine adjusted to pH to 2.8 is used as the mobile phase and 3μ particle size C18 column of 150 mm length and 4.6 mm internal diameter is used. Using photo diode array (PDA) detector, the compounds are monitored at 340 nm. All impurities are well separated and flow Gradient has been optimized to obtain the acceptable resolution between impurities and amlodipine. Diluent was chosen, based on the impurity peak shapes and recoveries. Test concentration and injection volume have been optimized to obtain limit of quantification (LOQ) values below the reporting threshold.

同时检测四种常见真菌毒素HPLC法的初步评价

真菌毒素是丝状产毒真菌生长过程中分泌的对人畜健康有害的次级代谢产物,广泛存在于多种农产品中。对已建立的同时检测黄曲霉毒素B1、玉米赤霉烯酮、赭曲霉毒素和桔霉素的方法,对其方法检出限、定量限、精密度和回收率进行了评价。该方法对上述4种真菌毒素的检出限分别为2.12、5.46、2.39和53.10μg/kg,定量限分别为8.48、21.82、9.57和212.38μg/kg;加标回收试验中4种毒素的回收率分别为81.43%,75.19%,80.44%和98.54%。该法简单、快速、高效,适合于批量样品分析,具有应用推广前景。

PRiME HLB固相萃取/超高效液相色谱-串联质谱法快速检测牛肝中18种促生长剂类药物残留

建立了PRiME HLB固相萃取/超高效液相色谱-串联质谱(UPLC-MS/MS)高通量快速检测牛肝中8种激素、6种β-受体激动剂及4种抗生素类促生长剂药物的方法。分别对色谱分离条件、MS/MS检测参数及样品前处理进行了优化。样品经β-葡萄糖醛苷酶/芳基硫酯酶酶解后,以甲醇和乙腈-甲醇(90∶10,体积比)分步提取后,直接通过PRiME HLB净化,收集流出液氮气吹干后以乙腈-水(3∶7,体积比)复溶,供UPLC-MS/MS检测。结果表明18种化合物在1.0~100.0μg/L范围内线性关系良好,相关系数均大于0.990;方法检出限(LOD)为0.07~0.78μg/kg,定量下限(LOQ)为0.23~2.58μg/kg。在3个加标水平下(0.5、1.0、5.0μg/kg)的回收率为67.5%~102.0%,相对标准偏差(RSD)均小于15%。该方法简单、快速、准确,适用于动物组织中多种促生长剂类药物的同时检测。

蔬菜中农药的使用对食品安全问题的影响

随着人们健康饮食意识的提高,蔬菜作为餐桌上的宠儿,因为其含有多种人身体所需的维生素及纤维素成分,越来越受到广大消费者的喜爱,蔬菜消费在中国居民的食品消费中占据极其重要的地位,但同时也存在一定的问题,那就是大家现在普遍关注的蔬菜携带农药的问题。农药就像一把双刃剑:一方面,可以帮助蔬菜作物除虫除害健康成长;另一方面,如果使用不当,会严重影响蔬菜的质量安全,并且蔬菜上这些残留的农药对人体也存在着潜在的危害。因此,蔬菜中农药残留问题引起了人们的高度重视。本文通过对蔬菜中农药残留状况的研究,对蔬菜中农药残留风险进行评估,以期为消费者、食品安全监管部门和蔬菜种植户们提供科学依据,促进我国蔬菜产业健康安全发展。

Toward a rapid and convenient nanoplastic quantification method in laboratory-scale study based on fluorescence intensity

The thorough investigation of nanoplastics(NPs)in aqueous environments requires efficient and expeditious quantitative analytical methods that are sensitive to environmentally relevant NP concentrations and convenient to employ.Optical analysis-based quantitative methods have been acknowledged as effective and rapid approaches for quantifying NP concentrations in laboratory-scale studies.Herein,we compared three commonly used optical response indicators,namely fluorescence intensity(FI),ultraviolet absorbance,and turbidity,to assess their performance in quantifying NPs.Furthermore,orthogonal experiments were conducted to evaluate the influence of various water quality parameters on the preferred indicator-based quantification method.The results revealed that FI exhibits the highest correlation coefficient(>0.99)with NP concentration.Notably,the limit of quantification(LOQ)for various types of NPs is exceptionally low,ranging from 0.0089 to 0.0584 mg/L in ultrapure water,well below environmentally relevant concentrations.Despite variations in water quality parameters such as pH,salinity,suspended solids(SS),and humic acid,a robust relationship between detectable FI and NP concentration was identified.However,an increased matrix,especially SS in water samples,results in an enhanced LOQ for NPs.Nevertheless,the quantitative method remains applicable in real water bodies,especially in drinking water,with NP LOQ as low as 0.0157–0.0711 mg/L.This exceeds the previously reported detectable concentration for 100 nm NPs at 40µg/mL using surface-enhanced Raman spectroscopy.This study confirms the potential of FI as a reliable indicator for the rapid quantification of NPs in aqueous environments,offering substantial advantages in terms of both convenience and cost-effectiveness.

气相色谱联用质谱法同时检测人体尿液中草甘膦及氨甲基膦酸含量

目的建立一种同时检测人尿样中草甘膦（PMG）和氨甲基膦酸（AMPA）含量的气相色谱联用质谱（GC/MS）分析方法。方法采用GC/MS法,EI检测器,色谱柱为DB-5MS毛细管色谱柱。进样前尿样经三氟乙酸酐（TFAA）和七氟丁醇（HFB）衍生化后用氮气吹干,0.2%柠檬醛乙酸乙酯溶液溶解混匀后供GC/MS分析。结果 PMG和AMPA在10~400 ng/ml范围内呈线性关系,最低检测限分别为4.66 ng/ml和1.77 ng/ml,最小检测浓度（LOQ）分别为15.52 ng/ml、5.91 ng/ml,回收率分别为85.2%~97.5%、89.2%~99.1%,精密度均小于10%。结论本实验可同时检测尿液中草甘膦及其代谢产物氨甲基膦酸,且最低检测限较单个检测方法灵敏。

高效液相色谱法测定苯嘧磺草胺颗粒剂中有效成分含量

[目的]研究苯嘧磺草胺颗粒剂中有效成分含量的定量分析方法。[方法]采用高效液相色谱法,使用C18柱,以乙腈-三氟乙酸水溶液为流动相,在254 nm波长下进行测定。[结果]方法的线性相关系数为1.0000,定量检测限(LOQ)为61.4 mg/L,最佳称量范围0.01～0.25 g,标准偏差为0.077 1,变异系数为0.396%,回收率为99.9%～102.7%。[结论]方法快速、准确,适用于苯嘧磺草胺颗粒剂的定量分析。

药品分析方法验证要点的讨论

分析方法验证是一个和分析方法建立相联系的过程,用于证明建立的方法及其参数满足分析应用要求。本文以《中华人民共和国药典》2015年版四部通则(9101)的验证内容为主线,对方法学验证中的一些要点进行了讨论。

GC-MS法测定奥硝唑注射液中的二甘醇

目的采用气相色谱-质谱联用法(GC-MS)测定奥硝唑注射液中二甘醇的含量。方法用乙醇溶解稀释样品,采用ZB-WAXplus;色谱柱(30 m×0.25 mm, 0.25μm),进样口温度为230℃,分流进样,分流比10:1,载气为高纯氦气(He),进样口为恒流模式,进样量为2μL,采用程序升温(初始温度40℃,保持2 min,先以30℃·min^(-1)的速率升温至160℃,保持2 min^(-1)再以10℃·min^(-1)的速率升温至210℃,保持3 min^(-1)最后以20℃·min^(-1)的速率升温至230℃,保持8 min),离子源为电子轰击源(EI),电离电压70 eV,离子源温度200℃,质谱传输接口温度240℃,质谱监测模式为选择离子(SIM),检测离子为m/z 45、m/z 75、m/z 76,溶剂延迟为9 min。按外标法定量。结果 11.74~187.82 ng·mL^(-1)二甘醇与峰面积的线性良好(r=0.9996);检出限为5.87 ng·mL^(-1),定量限为11.74 ng·mL^(-1)。仪器的精密度和方法重复性的RSD均<10%。平均回收率为96.4%,RSD=4.49%(n=9)。3批样品中二甘醇的含量(以奥硝唑的标示量计)分别为0.00002%、0.00004%、0.00001%,均小于定量限。结论所用方法简单、高效、重复性好,可用于检测奥硝唑注射液中二甘醇的含量。