Rapid and Sensitive Method to Detect Oxidized Forms of rhGCSF Using Agilent 2100 Bioanalyzer

Abstract

Oxidation of methionine of human granulocyte colony stimulating factor (GCSF) results in loss of biological activity. In this study, we report the use of an Agilent 2100 bioanalyzer to detect oxidized forms of rhGCSF after exposure to hydrogen peroxide (H₂O₂). Our data show that the bioanalyzer is capable of detecting minor changes in rhGCSF after oxidation with 0.01% (w/v) H₂O₂, which results in nearly 50% loss in biological activity as observed by cell (NFS-60) proliferation assay. Dithiothreitol could largely protect such a H₂O₂-mediated oxidation, and thus, we conclude that the major modification of GCSF upon methionine oxidation is the conversion of methionines to its sulfoxide.     

Method validation for the analysis of pesticide residues in grain by thin-layer chromatography

Method validation is an important requirement in the practice of pesticide residue analysis and is the process of verifying that a method is fit for its purpose. To make a correct decision on the validity of the method, the following method performance parameters have to be taken into consideration: scope, specificity, limit of detection, limit of quantification, linear range, accuracy, precision, repeatability, reproducibility, recovery, ruggedness and robustness. The goal of this study was to validate previously adapted thin-layer chromatographic methods for the pesticide residue analysis in grain. Confirmation of validation parameters for some compounds was also performed by gas chromatographic analysis.     

An overview of method validation and system suitability aspects in capillary electrophoresis

Summary Although capillary electrophoresis (CE) methods have been included in some regulatory submissions, the formal requirements for validation of such methods are not well-defined or universally understood. This paper provides a general overview of the aspects of method performance which need to be considered during the validation of CE methods particularly emphasising areas of similarity with, and differences between, HPLC and CE. The individual aspects of method performance are reviewed and guidance given, with illustrative literature examples where possible, on how to carry out suitable validation studies. Performance criteria are also provided, wherever possible. In this way, a suggested validation approach for CE methods is presented, reflecting the similarity with that generally adopted for HPLC methods, but accommodating those aspects which are different for, or peculiar to, CE.     

Method validation approach on the basis of a quadratic regression model

A method validation approach that bases on a quadratic regression model in which two types of error are incorporated is presented and applied to an experimental data set. The validation approach enables the determination of analytical performance characteristics referred to in Commission Decision 2002/657/EC (i.e., repeatability, within-laboratory reproducibility, decision limit, detection capability).     

Method validation of modern analytical techniques

 Validation of analytical methods of well-characterised systems, such as are found in the pharmaceutical industry, is based on a series of experimental procedures to establish: selectivity, sensitivity, repeatability, reproducibility, linearity of calibration, detection limit and limit of determination, and robustness. It is argued that these headings become more difficult to apply as the complexity of the analysis increases. Analysis of environmental samples is given as an example. Modern methods of analysis that use arrays of sensors challenge validation. The output may be a classification rather than a concentration of analyte, it may have been established by imprecise methods such as the responses of human taste panels, and the state space of possible responses is too large to cover in any experimental-design procedure. Moreover the process of data analysis may be done by non-linear methods such as neural networks. Validation of systems that rely on computer software is well established. The combination of software validation with validation of the analytical responses of the hardware is the challenge for the analytical chemist. As with validation of automated equipment such as programmable logic controllers in the synthesis of pharmaceuticals, method developers may need to concentrate on the process of validation, as well as the minutiae of what is done.     

Development and validation of a liquid chromatography method for analysis of colistin sulphate

Summary The peptide antibiotic Colistin has been successfully separated into more than thirty components on a YMC-Pack Pro, C-18 column with a mobile phase of acetonitrile—sodium sulphate (0.7% m/v)—phosphoric acid (6.8% v/v dilution of 85% m/m H₃PO₄)—water (21.5∶50∶5∶23.5) at 1.0 mL per min and detection at 215 nm. Baseline resolution of the major components, colistin A and B, and many minor components was obtained. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

Pressurised liquid extraction of flavonoids in onions. Method development and validation

A rapid and reliable analytical method for quantification of flavonoids in onions was developed and validated. Five extraction methods were tested on freeze-dried onions and subsequently high performance liquid chromatography (HPLC) with UV detection was used for quantification of seven flavonoids.The extraction efficiencies were lowest for the conventional water bath extraction compared to pressurized liquid extraction (PLE), ultrasonication, ultrasonic liquid processor, and microwave extraction, which yielded similar efficiencies. The reproducibility was in the same range (RSD: 1-11%) for all tested extraction methods. However, PLE was the preferred extraction method because the method can be highly automated, use only small amounts of solvents, provide the cleanest extracts, and allow the extraction of light and oxygen-sensitive flavonoids to be carried out in an inert atmosphere protected from light.The method parameters: extraction temperature, sample weight, flush volume and solvent type were optimised, and a clean-up step was integrated in the PLE procedure by in-cell addition of C18-material to the extraction cells, which slightly improved the recovery and reproducibility of the method. The one-step PLE method showed good selectivity, precision (RSDs = 3.1-11%) and recovery of the extractable flavonoids (98-99%). The method also appeared to be a multi-method, i.e. generally applicable to, e.g. phenolic acids in potatoes and carrots.     

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump‐free negative pressure sampling device was used for rapid bias‐free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025～4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 μg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5～6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump‐free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.     

Use of the Eurachem guide on method validation for determination of uranium in environmental samples

Three analytical methods for determination of uranium in environmental samples by a fluorescence technique have been validated and compared in accordance with the Eurachem Guide on method validation. The first method depends on uranium separation from iron using the mercury anode technique; in the other two methods uranium is separated from iron on an anion exchange column by use of either a solution of hydrochloric acid containing ascorbic acid and hydrazine hydrate or a dilute sulfuric acid solution. Detection limits, repeatability, reproducibility, and recovery coefficient were the main validation characteristics. The results showed that better statistical values can be achieved by using the third method. Control charts for in-house control samples and international intercomparison samples have also shown that the third method is more statistically stable with time. In addition, uncertainties of measurement were estimated and compared for the three methods. It was found that the Eurachem Guide and comparison of quality statistical validation data can be good tools for selection of the appropriate method for an application.     

Development and validation of a normal-phase high-performance thin layer chromatographic method for the analysis of sulfamethoxazole and trimethoprim in co-trimoxazole tablets

Pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals, such as HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of sulfamethoxazole (SMX) and trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets within resource-limited countries, Medicines Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets. SMX and TMP were separated on glass-backed silica gel 60 F₂₅₄ plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase was comprised of toluene, ethylacetate and methanol (50:28.5:21.5, v:v:v). Detection wavelength was 254 nm. The R_f values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. Cochran's criterion test indicated homoscedasticity of variances for the calibration data. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time-different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00% ± 1.83 and 99.66% ± 1.21 for SMX and TMP, respectively. The method was found to be robust and was then successfully applied to analyze co-trimoxazole tablet samples.     

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 °C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg²⁺ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.     

Degradation of polyurethanes in vitro and in vitro: comparison of different models

This study compares and contrasts mechanisms of polyetherurethane (PEU) degradation in vitro and in vivo. Models comprising incubation with hydrogen peroxide in vitro (H₂O₂), in vivo subcutaneous rat implant (SUBQ), and subcutaneous rat cage implant (CAGE) are described and compared with in vivo degradation of the pacemaker lead device retrieved after human implant (PACE). Experimental results support the hypothesis that stress accelerates PEU degradation. Scanning electron microscopy (SEM), gel permeation chromatography (GPC), and Fourier transform IR spectroscopy/attenuated total reflectance (FT-1R/ATR) evaluation of tested PEU samples suggests, for all models, decreased soft segment and increased ester functionality at the polymer surface. These observations are consistent with a single, metal ion catalyzed, polyester intermediate, oxidative degradation mechanism common to all models, and with device performance in vivo. Model comparison suggests that in vitro H₂O₂ and in vivo SUBQ and CAGE models accurately mimic in vivo degradation of the pacemaker lead device (PACE).     

Method validation of microcystins in water and tissue by enhanced liquid chromatography tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method has been developed and validated to identify and quantify trace levels of cyanotoxins or microcystins (MC) in water, bivalves and fish tissue with enhanced sensitivity and specificity. The method enables confirmation and quantification of six MCs (MC-LA, LF, LR, LW, RR and YR) with a single chromatographic run. The applied chromatography also allows determination of certain MC metabolites (Desmethyl-LR and -RR). By using LC-ESI–MS/MS in multiple reaction monitoring (MRM) mode, the limit of detection and quantitation for the microcystins studied, were determined to be between 0.2 and 1 pg on column (5:1 S/N ratio). These values are below the 2 pg detection limits found in the available literature.     

Usefulness of capability indices in the framework of analytical methods validation

Analytical methods capability evaluation can be a useful methodology to assess the fitness of purpose of these methods for their future routine application. However, care on how to compute the capability indices have to be made. Indeed, the commonly used formulas to compute capability indices such as Cpk, will highly overestimate the true capability of the methods. Especially during methods validation or transfer, there are only few experiments performed and, using in these situations the commonly applied capability indices to declare a method as valid or as transferable to a receiving laboratory will conduct to inadequate decisions.     

Method validation for direct determination of some trace and toxic elements in soft drinks by inductively coupled plasma mass spectrometry

The consumption of soft drink beverages has increased in the last few years around the world and it is related to the diversity of brands and flavours available, increasing also the risk of ingestion of compounds considered non-beneficial to the health of consumers. In this study, fast, easy and simple method of analysis for direct determination of As, Pb, Cd, Sb, Hg, Cu, Zn, Fe, Al, Cr, Sn, Co, Mn and Ni in soft drink samples using quadrupole inductively coupled plasma mass spectrometry (Q-ICP–MS) was validated. The estimated detection limits, practical quantification limits, linearity (linear dynamic ranges and method linearity), accuracy (trueness and precision) and measurement uncertainty parameters were studied under optimised (Q-ICP–MS) conditions. The method showed that the estimated detection limits were varied between 0.02 and 2.403 µg/L, and the quantification limits were varied between 0.5 and 20 µg/L. The mean recoveries ± standard deviations at different spiking levels were varied between 75.03 ± 0.62% and 117.07 ± 2.83% and the coefficients of variation were varied between 0.49% and 9.79%. The method trueness was confirmed by using four different certified reference materials (soft drinks and treated water) purchased from FAPAS (Food Analysis Performance Assessment Scheme) and all obtained results were within satisfactory ranges and had acceptable recovery and Z-score values. The method precision, in terms of relative standard deviation, was below 4.88%. The method uncertainty expressed as expanded uncertainty of all validated elements was found to be ≤22.52%. The results obtained make the method suitable for accurate determination of validated elements in different kinds of soft drink samples at these low concentration values. Validated method was used for the determination of metallic contaminants in 40 commercial soft drink samples and the results were compared with the provisional guideline of the elements stated by Egyptian, WHO and European standards in drinking and potable bottled natural mineral water.     

Method validation in quantitative electrochemical analysis of colchicine using glassy carbon electrode

A cathodic differential pulse voltammetric determination of colchicine was validated using a glassy carbon electrode in HClO₄/H₃PO₄ 0.01 M. Colchicine gives an irreversible, diffusion-controlled peak at −862 mV vs. Ag/AgCl reference electrode. The cathodic peak is strongly influenced by a more alkaline environment with a shift towards more negative potentials. Method optimization was carried out in parallel for three types of electrodes (glassy carbon, mercury film and bismuth film coated glassy carbon). The cathodic peak current is higher using film-coated electrodes, but shows poorer intra-day reproducibility and a longer analysis time due to film renewal. Thus, a bare glassy carbon electrode was used to determine colchicine in the concentration range of 2.4 − 50 μg mL⁻¹ (R ² = 0.9998, n = 5), with a calculated detection limit of 0.80 μg mL⁻¹. The proposed method was characterized according to ICH Harmonized Tripartite Guidance Q2(R1) by validation parameters (selectivity, linearity, accuracy, fidelity, limit of detection, limit of quantification) and it was successfully applied for the determination of colchicine from tablets, without the interference of the excipients. The method’s performances were evaluated and compared with both a known polarographic method and the official quantitative spectrophotometric determination from the Romanian Pharmacopoeia, Xth edition, respectively.      

A tutorial on the validation of qualitative methods: From the univariate to the multivariate approach

This tutorial provides an overview of the validation of qualitative analytical methods, with particular focus on their main performance parameters, for both univariate and multivariate methods. We discuss specific parameters (sensitivity, specificity, false positive and false negative rates), global parameters (efficiency, Youden's index and likelihood ratio) and those parameters that have a quantitative connotation since they are usually associated to concentration values (decision limit, detection capability and unreliability region).     

Optimization and validation of stability indicating RP-HPLC method for the quantification of gefitinib in bulk drug and nanoformulations: An application towards in vitro and ex vivo performance evaluation

Gefitinib (GFB) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used primarily to treat non-small cell lung cancer (NSCLC), but it also works by lowering AKT and MAPK phosphorylation, which makes it effective against the breast cancer cells. A high-performance liquid chromatography (HPLC) method was developed for detecting gefitinib by C18 analytical column with mobile phase containing acetonitrile and 1 % w/v ammonium acetate in water with ratio of 60:40. Box-Behnken design was used to optimize the chromatographic conditions, and method was validated for accuracy, precision, linearity, robustness, ruggedness, limit of detection, and limit of quantification. The developed method was found to be useful in estimating gefitinib in conventional marketed tablet dosage forms and nanoformulations such as SLNs and liposomes. With a calibration curve, linearity was attained in the drug concentration range of 8–56 µg/mL (r² = 0.9996), and sensitivity was found to be 1.3 and 3.9 µg/mLas LOD and LOQ, respectively. All the validation criteria for the method were within the acceptable limits. The drug content in the conventional tablet formulation was found to be 99.99 %. The HPLC analysis indicated that gefitinib was highly soluble in Precirol ATO5 as solid lipid and tween 80 as surfactant. Drug entrapment efficiency was found to be 83.1 % and 80.5 % for SLNs and liposomes respectively. In vitro release data revealed that 30 % of drug was released from plain suspension and more than 77 % released from both nanoformulations after 24 h at pH 7.4 respectively. By applying kinetic fit, data was most appropriately fitted into first order as compared to other. The apparent permeability of gefitinib from plain suspension, SLNs and Liposomes was found to be 3.98 × 10^?4 cm/min, 1.35 × 10^?4 cm/min and 1.79 × 10^?4 cm/min.     

Method validation for determination of arsenic, cadmium, chromium and lead in milk by means of dynamic reaction cell inductively coupled plasma mass spectrometry

With Regulation No. 1881/2006 the European Union fixed a maximum level for lead in milk. Consequently, there is the need to determine very low concentration of elements that may be present in milk in trace and ultratrace levels.Quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) combined with dynamic reaction cell (DRC) has been widely employed in order to reach very low concentration, requested for this product. Furthermore, the DRC technology can help in removing polyatomic and argon-based interferences.In the present study, a method for the determination of arsenic, cadmium, chromium and lead in bovine milk was validated according to the EU common standards by means of DRC-ICP-MS. The main parameters evaluated in the validation were: recovery, repeatability and within-laboratory reproducibility, detection and quantification limits, linearity range and measurement uncertainty. Additionally, stability studies of the analyte in solution and ruggedness studies were carried out.The results obtained for limit of detection (LoD) and limit of quantification (LoQ) in μg kg⁻¹ were respectively: As, 3.1 and 9.5; Cd, 0.08 and 0.24; Cr, 0.229 and 0.693; Pb, 0.5 and 1.5. While for the recovery: As, 91%; Cd 96%; Cr 99%; Pb, 95%. As for the repeatability: As, 7%; Cd, 3%; Cr, 6%; Pb, 4%.