Ion-pairing HPLC methods to determine EDTA and DTPA in small molecule and biological pharmaceutical formulations

Ion-pairing high-performance liquid chromatography-ultraviolet （HPLC-UV） methods were developed to determine two commonly used chelating agents, ethylenediaminetetraacetic acid （EDTA） in Abilify （a small molecule drug with aripiprazole as the active pharmaceutical ingredient） oral solution and die- thylenetriaminepentaacetic acid （DTPA） in Yervoy （a monoclonal antibody drug with ipilimumab as the active pharmaceutical ingredient） intravenous formulation. Since the analytes, EDTA and DTPA, do not contain chromophores, transition metal ions （Cu2＋, Fe3＋） which generate highly stable metallocom- plexes with the chelating agents were added into the sample preparation to enhance UV detection. The use of metallocomplexes with ion-pairing chromatography provides the ability to achieve the desired sensitivity and selectivity in the development of the method. Specifically, the sample preparation in- volving metallocomplex formation allowed sensitive UV detection. Copper was utilized for the de- termination of EDTA and iron was utilized for the determination of DTPA. In the case of EDTA, a gradient mobile phase separated the components of the formulation from the analyte. In the method for DTPA, the active drug substance, ipilimumab, was eluted in the void. In addition, the optimization of the concentration of the ion-pairing reagent was discussed as a means of enhancing the retention of the aminopolycarboxylic acids （APCAs） including EDTA and DTPA and the specificity of the method. The analytical method development was designed based on the chromatographic properties of the analytes, the nature of the sample matrix and the intended purpose of the method. Validation data were presented for the two methods. Finally, both methods were successfully utilized in determining the fate of the chelates.

An Ion-Pair HPLC Method for Simultaneous Determination of Exogenous Phosphocreatine and Its Metabolite Creatine and Related ATP in Rabbit Plasma and RBC: Application to a Pharmacokinetic Study

A specific, precise and accurate ion-pair HPLC-UV method has been developed and validated for simultaneous determination of phosphocreatine (PCr), and its metabolite creatine (Cr) as well as related ATP in plasma and red blood cell (RBC) of rabbits. After addition of TMP as IS, the samples were deproteinized with 6% PCA. The analytes were separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of buffer A (0.2% KH2PO4 + 0.08% tetrabutyl ammonium hydrogen sulphate, pH 3.0), buffer B (buffer A adjusted to pH 7.5 with 1 mol/L NaOH) and MeOH. Detection wavelengths were set at 210 nm for PCr and Cr and 260 nm for ATP and TMP. Some blank samples were initially run for baseline subtraction. The linear detection responses were obtained for PCr concentration over a range of 10 - 7500 mg/ml (plasma) and 5 - 2500 mg/ml (RBC) and for both Cr and ATP concentrations of 10 - 1500 mg/ml (plasma) and 5 - 750 mg/ml (RBC) (r > 0.99). The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of - 107%. The method was successfully used to simultaneously determine plasma and RBC concentrations of the 3 analytes and to study pharmacokinetics after iv administration of PCr to rabbits.

A HPLC Method for the Determination of Salmon Calcitonin in Injection by Gradient Elution

It was concluded that the described HPLC method could be used for the assayof salmon calcitonin in injection, as it offers qualified selectivity, accuracy and precision ofanalysis.

Development of an HPLC–UV assay method for the simultaneous quantification of nine antiretroviral agents in the plasma of HIV-infected patients

A new method using high-performance liquid chromatography coupled with ultra violet detection(HPLC–UV)was developed and validated for the simultaneous quantification of atazanavir,dolutegravir,darunavir,efavirenz,etravirine lopinavir,raltegravir,rilpivirine and tipranavir in human plasma.For the first time we reported here the development and validation of an HPLC–UV assay to quantify the frequently administered 9antiretroviral compounds including dolutegravir and rilpivirine.A simple solid phase extraction procedure was applied to 500 μL aliquots of plasma.The chromatographic separation of the drugs and internal standard(quinoxaline) was achieved with a gradient of acetonitrile and sodium acetate buffer on a C_(18) reverse-phase analytical column with a 25 min analytical run time.Calibration curves were optimised according to the therapeutic range of drug concentrations in patients,and the coefficient of determination(r^2) was higher than0.99 for all analytes.Mean intraday and interday precisions(RSD) for all compounds were less than 15.0%,and the mean accuracy(% deviation from nominal concentration) was also found to be less than 15.0%.Extraction recovery range was between 80% and 120% for all drugs analysed.The solid phase extraction and HPLC–UV method enable a specific,sensitive,and reliable simultaneous determination of nine antiretroviral agents in plasma.Good extraction efficiency and low limit of HPLC–UV quantification make this method suitable for use in clinical trials and therapeutic drug monitoring.

基于HPLC-Q-TOF-MS/MS技术的半夏姜制前后成分分析

目的 采用高效液相色谱-四极杆飞行时间质谱法(HPLC-Q-TOF-MS/MS)对半夏及姜半夏的成分进行鉴定,并比较姜制前后化学成分差异。方法 采用HPLC-Q-TOF-MS/MS技术,对半夏和姜半夏的成分进行检测。将采集得到的样品数据导入质谱数据分析软件Agilent MassHunter Qualitative Analysis 10.0,对各个质谱峰进行提取。搜索自建的数据库,初步鉴定化学成分。再结合文献中化合物、对照品以及MassBank数据库中相关成分的碎片离子信息和保留时间,进行成分确定。此外,将半夏和姜半夏的质谱数据导入安捷伦Profinder 10.0软件,对数据进行峰提取、对齐、归一化等处理。利用Simca 14.1软件对数据进行主成分分析(PCA)及正交偏最小二乘法判别分析(OPLS-DA)。结果 在半夏及姜半夏中共鉴定得到43个成分,其中生物碱类成分5个,脂肪酸甘油酯类成分7个,溶血磷脂胆碱类成分7个,醇胺类成分5个,氨基酸类成分7个,酰胺类成分5个,有机酸类1个,姜辣素类2个,其他类成分4个。PCA分析结果显示,半夏姜制前后化学成分具有较大差异。OPLS-DA结果表明,通过VIP值>1,|P(corr)|>0.5筛选得到腺苷、鸟苷、丝胶树碱等18个主要差异成分。结论 半夏炮制前后,部分生物碱类、酰胺类、姜辣素类等成分存在明显差异,这些差异性化合物可能导致了半夏姜制前后功效的侧重不同,为提升半夏及其姜制品的质量标准提供参考。

HPLC Determination of the Major Non-protein Amino Acids and Common Biogenic Amines in Lathyrus sativus Using a Novel Extraction Method

An assay is presented for simultaneously determining 5 biogenic amines and the major non-protein amino acids: the toxin β-N-oxalyl-L-α,β-diaminopropanoic acid (β-ODAP), its isomer α-ODAP and homoarginine in Lathyrus sativus extracts using the HPLC system after derivatization with para-nitrobenzyloxycarbonyl chloride (PNZ-Cl). However, it is more worthy of noting that this paper also describes a new extraction method using 0.2 mol/L HClO4. The new method has some advantages: shorter extraction-time, simultaneous extraction of free amino acids and polyamines, better inhibiting the isomerization of β-ODAP to α-ODAP, and so on.

RP-HPLC Method for the Simultaneous Determination of Lisinopril and NSAIDs in API, Pharmaceutical Formulations and Human Serum

High performance liquid chromatographic method was developed valdated and applied for the simultaneous determi- nation of lisinopril and NSAIDs in bulk, pharmaceuticals formulations and human serum. A Purospher star C18 (5 μm, 25 × 0.46 cm) column was used with mobile phase consisting of methanol: water: acetonitrile (80:17.5:2.5 v/v, pH 3.0) and quantitative evaluation was performed at 225 nm with a flow rate of 1.0 mL?min–1. The retention time of lisinopril was 2.2 min while naproxen, flurbiprofen, diclofenac sodium and mefenamic acid were found to be 4.0, 4.5, 5.0 and 6.7 min respectively. Suitability of this method for the quantitative determination of the drugs was proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines. The method is selective, precise, accurate and can be used for analysis of pharmaceutical preparations in quality control and clinical laboratories.

The Quantum Scattering Study for Ion-pair Formation Reaction Na+I_2→Na^+I_2^- with the LCAC-SW method

The selected-state probabilities of collinear ion-pair formation process Na+I2→Na++I2-on Aten-Laming-Los two-State potential energy surface have been calculated by using LCAC-SW method. The results show that reaction probabilities are oscillatory with collision energy; the threshold energy of this ioniZation reaction is 2.8 ev, which is in modest agreement with experimental result.

Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue

The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.

Development of simple HPLC/UV with a column-switching method for the determination of nicotine and cotinine in hair samples

Nicotine and cotinine in hair are good biomarkers for assessing long-term exposure to smoking. However, analytical devices such as GC/MS are associated with high cost and are not widely used. HPLC/UV is used widely in laboratories, but is unsuitable for measurement of minor constituents, except when using the column-switching method. Thus, we aimed to establish a simple, inexpensive and sensitive method based on HPLC/UV with column switching for measuring nicotine and cotinine in hair. First, we compared the presence and absence of a column selection unit. We then measured amounts of nicotine and cotinine in hair samples collected from the general population, and compared both the corresponding levels and the detection limits with those in previous studies. Finally, initial and running costs of HPLC/UV were compared with other analytical methods. As one of the results, the areas of nicotine and cotinine measured by HPLC/UV with column-switching method were 12.9 and 16.9 times greater, respectively, than those without the column-switching method. The amount of nicotine and cotinine in hair was significantly correlated to number of cigarettes smoked per day (r = 0.228, p = 0.040). In addition, the HPLC/UV method showed similar sensitivity and detection limit (nicotine, 0.10 ng/mg;cotinine, 0.08 ng/mg) as reported in previous studies. The cost of the HPLC/UV method is lower than that of other analytical methods. We were able to establish a low-cost method with good sensitivity for measuring nicotine and cotinine in hair. The HPLC/UV with a column-switching method will be useful as a first step in screening surveys in order to better understand the effects of smoking exposure.

New HPLC Method with Experimental Design and Fluorescence Detection for Analytical Study of Antihypertensive Mixture,Amlodipine and Valsartan

New HPLC method was developed for determination of amlodipine and valsartan in their binary mixture as a part of routine control of combined formulations. The method was validated to meet official requirements including selectivity, stability, linearity, precision and accuracy. Chromatography was carried out using a LiChrospher RP-18 column, a mixture containing acetonitrile, phosphate buffer of pH 3.5 and methanol (45:45:10, v/v/v) and new fluorescence detection at 255 nm for excitation and 448 nm for emission. The effect of methanol content, pH of the buffer, flow rate, detection wavelengths and column temperature was estimated in robustness study, according to a plan defined by the Plackett-Burman design. For identification of significant effects, both graphical and statistical methods were used. Ro-bustness for dissolution test was checked estimating the effects of paddle speed, temperature and pH of dissolution medium. The method was proved to complying with all official guidelines. Therefore, it is suitable for determination of amlodipine and valsartan in their binary mixtures for different analytical and pharmaceutical purposes.

Determination of Fenofibrate and the Degradation Product Using Simultaneous UV-Derivative Spectrometric Method and HPLC

Two new selective, precise, and accurate methods were developed for the determination of fenofibrate in the presence of its basic degradation product. In the first method fenofibrate was determined using an algorithm bivariate calibration derivative method, in which an optimum pair of wavelengths was chosen for the determination of different binary mixtures. In the second method (HPLC), separation was achieved on RESTEK Pinnacle II phenyl column (5 μm, 250 × 4.6 mm) and Pinnacle II phenyl (5 μm, 10 × 4 mm) guard cartridge using a mobile phase consisting of methanol –0.1% phosphoric acid (60:40, v/v) at a flow rate 2 mL●min–1, and the column oven temperature was set at 50°C. The UV detector was time programmed at 302 nm and 289 nm for the internal standard (I.S.) and fenofibrate, respectively. The proposed methods were successfully applied for the determination of fenofibrate and its degradation product in the laboratory-prepared mixture and in pharmaceutical formulation. The assay results obtained using the bivariate method were statistically compared to those of the HPLC method and good agreement was observed.

Development and Application of a Validated HPLC Method for the Determination of Clindamycin Palmitate Hydrochloride in Marketed Drug Products: An Optimization of the Current USP Methodology for Assay

A simple efficient isocratic reversed-phase HPLC method was developed and validated for the determination of clindamycin palmitate hydrochloride (CPH) and its commercially available oral solution products. Separation was achieved on a Phenomenex Zorbax (Luna) cyano column (150 × 4.6 mm, 5 μm) with a Phenomenex cyano guard cartridge (4 × 3.0 mm) on Agilent 1050 series HPLC system. CPH and its resolution standard lincomycin were eluted isocratically at a flow rate of 1 mL/min with a simplified mobile phase (potassium phosphate buffer (5 mM, pH 3.0)—acetonitrile—tetrahydrofuran (20:75:5, v/v/v)) and detected at 210 nm. The column was maintained at 25?C. The method was validated according to USP category I requirements. Robustness and forced degradation studies were also conducted. CPH marketed drug products were obtained from a drug distributor and assayed for potency using the validated method. Validation acceptance criteria were met in all cases. The analytical range for CPH was 15 - 500 μg/mL and the linearity was r2 > 0.999 over three days. The method was determined to be specific and robust. Both accuracy (92.0% - 103.8%) and precision (0.67% - 1.52%) were established across the analytical range for low, intermediate and high QC concentrations. Method applicability was demonstrated by analyzing two marketed products of CPH, in which results showed potency >98%. The method was determined to be an enhancement over the current USP methodology for assay as a result of increased efficiency, reduced organic solvents and the elimination of matrix modifiers. This method was successfully applied for the quality assessment of: 1) currently marketed drug products and 2) will in future assess the product quality of novel dosage forms of CPH for pediatric use.

Stability Indicating HPLC Method for Quantification of Solifenacin Succinate &Tamsulosin Hydrochloride along with Its Impurities in Tablet Dosage Form

A novel stability-indicating RP-HPLC method was developed and validated for simultaneous determination of Solifenacin Succinate & Tamsulosin Hydrochloride and its impurities in tablet dosage form. The method was developed using L1 column with gradient using the mobile phase consist of solvent-A (pH = 6.6, phosphate buffer + 0.5% Triethylamine) and solvent-B (90% Acetonitrile). The eluted compounds were monitored at 225 nm. Solifenacin Succinate & Tamsulosin Hydrochloride was subjected to oxidative, acid, base, hydrolytic, thermal and photolytic stress conditions. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantitation, accuracy, precision and robustness. The limit of quantification results was ranged from 0.135 - 0.221 μg/mL for Solifenacin Succinate impurities and 0.043 - 0.090 μg/mL for Tamsulosin Hydrochloride impurities. This method is suitable for the estimation of impurities and assay of Solifenacin Succinate & Tamsulosin Hydrochloride in tablets dosage form.

HPLC法测定栀子药材中栀子苷含量的能力验证研究

目的开展栀子药材中栀子苷含量测定的能力验证研究,评价药品相关领域检验检测实验室开展中药材中指标成分含量测定的能力,提高相关实验室含量测定的质控能力。方法依据CNAS-RL02《能力验证规则》和国际标准ISO/IEC 17043《合格评定能力验证的通用要求》进行实验室能力验证活动。根据CNAS-GL003《能力验证样品均匀性和稳定性评价指南》对自制样品均匀性和稳定性试验结果进行分析,试验结果合格后作为能力验证样品,随机分发给参加者,回收结果,并对测定结果进行稳健统计分析,以Z比分数对各实验室结果进行判定,评价结果分为满意、可疑、不满意。结果共有403家实验室提交试验结果,其中能力验证结果为满意的有367家,满意率为91.07%;可疑的有17家,占4.22%;不满意的有19家,不满意率为4.71%。结论403家实验室中,大多数具备HPLC法测定栀子中栀子苷含量的能力,其中药品监管系统实验室检测能力和质量管理水平较高。本次能力验证为了解我国药品检验检测实验室的技术储备能力、管理水平提供依据,为今后的政府监管提供技术支撑。

HPLC-UV法测定舒肝和胃丸中α-香附酮和香附烯酮的含量

目的:建立舒肝和胃丸中α-香附酮和香附烯酮的高效液相色谱(HPLC-UV)含量测定法,为该制剂中醋香附含量的质量控制提供依据。方法:对舒肝和胃丸中α-香附酮和香附烯酮同时进行含量测定。采用Agilent ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇和水(65∶35),流速1.0 mL·min^(-1),检测波长254 nm,柱温30℃,进样量5μL。结果:采用该方法得到的舒肝和胃丸色谱图中,α-香附酮和香附烯酮色谱峰与相邻色谱峰分离效果较好,满足含量测定的要求;指标性成分α-香附酮、香附烯酮分别在0.08036~0.80360μg、0.2112~2.1120μg范围内线性关系良好,相关系数均为1.000;平均加标回收率分别为102.0%(RSD:1.6%,n=6),102.4%(RSD:2.0%,n=6);稳定性(24 h)、重复性、精密度良好。结论:本研究建立的α-香附酮和香附烯酮HPLC-UV含量测定方法简单、快捷、准确,可为舒肝和胃丸中醋香附含量的控制提供依据。

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%.

HPLC法测定蔬菜中α-胡萝卜素和β-胡萝卜素的前处理方法优化

目的优化HPLC法测定蔬菜中α-胡萝卜素和β-胡萝卜素的前处理方法,以提高现有测定方法的检测效率。方法本研究以国标GB 5009.83-2016为基础,通过优化皂化条件、酸化法代替水洗法等,改进蔬菜中α-胡萝卜素和β-胡萝卜素测定的前处理方法。结果本研究利用酸化法改良后,方法的平均加标回收率为86.9%~98.7%,相对标准偏差为3.2%~5.4%,在0.5~10μg·mL^(-1)线性范围内线性良好。结论本研究通过优化HPLC的前处理方法,显著提高了蔬菜中胡萝卜素的检测效率,节省了人力与试剂成本,为胡萝卜素检测技术的改良提供了创新性的思路与方向,可广泛应用于蔬菜的品质检测与营养评估。

Stability Indicating RP-HPLC Method for Quantification of Impurities in Valsartan and Hydrochlorothiazide FDC Tablet Dosage Form

A stability-indicating RP-HPLC method has been developed and validated for simultaneous determination of Valsartan & Hydrochlorothiazide and their impurities in FDC (Fixed Dose Combination) tablet dosage form. The method was developed using L1 column (250 × 4.6 mm;5 μm) with gradient elution using the mobile phase consisting of solvent-A (0.1% Ortho phosphoric acid) and solvent-B (100% Acetonitrile);the gradient program (Tmin/%B) was set as 0/10, 5/10, 20/60, 40/60, 41/10 and 50/10. The eluted compounds were monitored at 265 nm. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantitation, accuracy, precision and robustness. The influence of Acid, Alkaline, Oxidative, Photolytic, Thermal and Humidity stress conditions, on drug product was studied. The limit of quantification results of Valsartan, Hydrochlorothiazide and their impurities are, VAL: 0.303 μg/mL, HCTZ: 0.019 μg/mL, VAL RC-B: 0.085 μg/mL, VAL RC-C: 0.327 μg/mL, HCT RC-A: 0.017 μg/mL, CTZ: 0.080 μg/mL and 5-Chloro HCT: 0.047 μg/mL. The proposed method is suitable for the estimation of Valsartan & Hydrochlorothiazide impurities in tablets dosage form.

Development and Validation of Stability Indicating RP-HPLC-PDA Method for Tenatoprazole and Its Application for Formulation Analysis and Dissolution Study

In the present study, comprehensive stress testing of tenatoprazole was carried out according to ICH guide-line Q1A (R2). Tenatoprazole was subjected to stress conditions of hydrolysis, oxidation, photolysis and neutral decomposition. Extensive degradation was found to occur in acidic, neutral and oxidative conditions. Mild degradation was observed in basic conditions. The drug is relatively stable in the solid-state. Successful separation of drug from degradation products formed under stress conditions was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5.0 μ particle size) using methanol: THF: acetate buffer (68:12:20 v/v) pH adjusted to 6.0 with acetic acid as mobile phase, flow rate was 1.0 mL●min–1 and column was maintained at 45°C. Quantification and linearity was achieved at 307 nm over the concentration range of 0.5 - 160 μg●mL–1 for tenatoprazole. The method was validated for specificity, linearity, accuracy, precision, LOD, LOQ and robustness.