Ion chromatographic method development for monitoring of seawater quality used in over-the-counter pharmaceutical industry

A methodology is developed for the analysis of inorganic anions (fluoride, chloride, bromide, sulphate) in seawater used for over-the-counter (OTC) nasal spray production. The eluent flow rate and concentration of eluent competing ions are optimised by using an artificial neural network resolution model in combination with normalised resolution product criterion function. The developed artificial neural network resolution model shows good predictive ability R2 > or = 0.9973. The determined ion chromatographic parameters enable baseline separation of all components of interest. By performing a validation procedure and a number of statistical tests, it is shown that the developed ion chromatographic method has superior performance characteristics: linearity R2 > or = 0.9993, recovery = 99.77-100.65%, repeatability RSD < or = 1.85%. This result proves that the proposed method can be used for routine quality assurance analysis in OTC pharmaceutical industry.     

Automated resolution of overlapping peaks in chromatographic data

Parallel factor analysis 2 (PARAFAC2) has been shown to be a powerful tool for resolution of complex overlapping peaks in chromatographic analyses. It is particularly useful because of its ability to handle shifts in the elution time mode and peak shape changes. Like all curve resolution techniques, PARAFAC2 will only find chemically meaningful parameters (elution time profiles and mass spectra) if the correct number of factors are determined. So far, the primary way to determine an appropriate number of factors, when using PARAFAC2, is to calculate models with different number of factors and then inspect the models manually. This approach is time consuming, and the result may be biased because of the manual assessment of the model quality, making PARAFAC2 inaccessible for analytical chemists in general. Here, we develop a method that can determine an appropriate number of factors in an automated way. The automation is based on a number of model diagnostics (quality criteria) collected from models with different numbers of factors. Combining these diagnostics, it is possible to assess what the appropriate number of components is. In this work, only gas chromatography–mass spectrometry data are considered. However, it will most likely be fairly straightforward to expand the work to also cover liquid chromatography data (with a multivariate detector). Automating the model quality evaluation of the PARAFAC2 model enables both the inexperienced and trained user to perform comprehensive and advanced analysis of chromatographic data with a minimum of manual work. © 2013 The Authors. Journal of Chemometrics Published by John Wiley & Sons Ltd.     

Validation of standardized high-performance thin-layer chromatographic methods for quality control and stability testing of herbals

In herbal medicinal products the entire herbal drug or an herbal drug preparation is regarded as the active pharmaceutical ingredient, regardless of whether constituents with defined therapeutic activity are known. In quality control and stability testing of herbal medicinal products, fingerprint chromatograms are used as powerful tools to evaluate and compare the composition of compounds in such products. To fulfill the International Conference on Harmonization and Good Manufacturing Practice-based regulatory requirements in pharmaceutical quality control, chromatographic fingerprint analysis needs to be validated. Based on a standardized methodology, this paper provides a comprehensive concept for evaluating validation parameters for planar chromatographic fingerprinting by considering the stationary phase, sample application, developing solvent, chromatogram development, plate labeling, derivatization, documentation, and chromatographic equipment. Validation parameters addressed include stability of the analyte, selectivity, robustness testing, and method reproducibility.     

The NIH analytical methods and reference materials program for dietary supplements

Quality of botanical products is a great uncertainty that consumers, clinicians, regulators, and researchers face. Definitions of quality abound, and include specifications for sanitation, adventitious agents (pesticides, metals, weeds), and content of natural chemicals. Because dietary supplements (DS) are often complex mixtures, they pose analytical challenges and method validation may be difficult. In response to product quality concerns and the need for validated and publicly available methods for DS analysis, the US Congress directed the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) to accelerate an ongoing methods validation process, and the Dietary Supplements Methods and Reference Materials Program was created. The program was constructed from stakeholder input and incorporates several federal procurement and granting mechanisms in a coordinated and interlocking framework. The framework facilitates validation of analytical methods, analytical standards, and reference materials.     

On-line microdialysis of proteins with high-salt buffers for direct coupling of electrospray ionization mass spectrometry and liquid chromatography

Mass spectrometry (MS) is one of the most powerful instrumental techniques for protein analysis. The electrospray ionization (ESI) approach is known to be very gentle and at the same time compatible with liquid separation techniques such as HPLC and CE. However, ESI is known to be susceptible to salts and impurities, which often cause a dramatic decrease in sensitivity due to the suppression of the ionization of the product of interest. For this reason, LC-ESI-MS coupling has so far been largely limited to reversed-phase chromatography with its hydro-organic mobile phases. Other chromatographic techniques are typically "linked" to ESI-MS by time consuming, off-line desalting steps. On-line microdialysis has been proposed as a solution to this dilemma. In this paper, we introduce an improved microdialysis system, which enlarges the number of putative applications, thus allowing chromatographic separations of biological compounds to be directly coupled to MS detection with little to no loss in time or chromatographic resolution. Examples include separations by affinity, ion-exchange and size-exclusion chromatography, all of which were connected successfully to the ESI-MS detector via the on-line microdialyzer. We propose that, using this system, any kind of chromatography technique can be coupled to ESI-MS, thus enabling for example application in quality control or process monitoring of many bioproduction and downstream processes.     

Multi-objective optimisation strategy based on desirability functions used for chromatographic separation and quantification of l-proline and organic acids in vinegar

An optimisation strategy based on desirability functions together with experimental design has been used to optimise a chromatographic method applied to the separation and quantification of l-proline and seven organic acids in vinegar samples. Chromatographic problems often force to reach a compromise between different experimental variables in order to achieve the best chromatographic separation. The importance of the use of multi-objective optimisation methods lies in the ability to cope with this kind of problems. The quality of the multicriteria optimisation method was tested through the validation of the analytical parameters of the final chromatographic method developed. The versatility of this methodology allows to use it in other chromatographic applications resulting in a suitable adaptive procedure to solve new analytical problems. Furthermore, the determination of l-proline and organic acids in vinegar is useful for several industrial goals such as the correct monitoring of fermentation, for the study of nutrient needs at all times during the twofold fermentation process and for the detection of possible adulterations in the final product.     

Development, optimization and validation of gas chromatographic fingerprinting of Brazilian commercial gasoline for quality control

Three-step development, optimization and validation strategy for GC fingerprints of Brazilian commercial gasoline is described. A suitable chromatographic system was selected first. The following step was to improve acceptable chromatographic resolution with reduced analysis time, which is recommended for routine applications. Optimization was carried out using full three-level factorial designs. Optimal conditions were obtained for flow rate, oven ramps, injection volume and split ratio. Finally, several validation parameters were performed. Therefore, a feasible and reliable fingerprint was established to identify Brazilian commercial gasoline quality. This strategy can also be applied to develop fingerprints for quality control of other fuels.     

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.     

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.     

Simple and Efficient Solution for Robustness Testing in Gradient Elution Liquid Chromatographic Methods

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.     

Estimating optimal time for fast chromatographic separations

The term t_{min cc} provides a ready estimate of the shortest time that can be obtained by “column cutting” for baseline resolution of two components showing excess chromatographic resolution. While actual column cutting is impractical, the t_{min cc} value is shown to be closely related to the minimum separation time obtainable by adjusting other parameters such as flow rate, mobile phase composition, and temperature, affording scientists interested in the development of fast chromatographic separations a convenient tool for estimating the minimum separation time that can be obtained by modifying a given method development screening result. Furthermore, the relationship between t_{min cc} and the minimum separation time obtainable by adjusting other parameters is shown to be dependent on the speed of the screening method, with aggressive screening gradients affording t_{min cc} estimates that match the actual minimum separation time, and “lazy” screening gradients affording t_{min cc} values that overestimate minimum separation time. Consequently, the analysis of the relationship between t_{min cc} and actual minimum separation time may be a useful tool for determining the “fitness” of method development screening methods.     

Resolution of Overlapping Chromatographic Peaks Using a Genetic Algorithm

ABSTRACT

A genetic algorithm for resolution of overlapping chromatographic peaks (GAROCP) using real-number coding, non-uniform mutation and arithmetical crossover methods is described in this paper. It was applied to resolution of highly overlapped multicomponent high-performance liquid chromatographic peaks by fitting experimental chromatogram to the exponentially modified Gaussian (EMG) model. The genetic algorithm was used to find the minimum of fitting error to optimize the parameters in the EMG functions which determine the shape and area of each peak. The applicability of the method was investigated with both simulated signals calculated by EMG functions and experimental multicomponent overlapping chromatograms.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation

The most important steps in the validation of high-performance liquid chromatographic (HPLC) methods are discussed. The establishment of system suitability data and the assessment of peak purity are demonstrated on the example of bisquaternary amino steroids. For the recognition of incomplete resolution of adjacent peak pairs, the absorbance-ratio method in which the ratio of absorbances at two preselected wavelengths are plotted as a function of time in combination with the separation of sample components subjected to various chemical and physico-chemical treatments (stress conditions) is applied. The separation power and performance of the HPLC systems are characterized by the system resolution (SR) and system selectivity (SS). The special demands of stability-indicating methods are summarized.     

Development of an ultraperformance liquid chromatography method for improved determination of additives in polymeric materials

An analytical method for the simultaneous separation and quantitation of ten colorants and six antioxidants used as polymer additives has been developed by transferring chromatographic conditions from two independent HPLC methods to a single ultraperformance liquid chromatography system. An experimental design was carried out to achieve the best elution gradient for separation of all the studied compounds with the best possible peak resolution and in the shortest possible analysis time. The new method gives good values for the analytical quality parameters evaluated.     

Characterization of high performance glass capillary gas chromatography

Summary High resolution gas chromatography requires the highest performance characteristics of gas chromatographic systems in terms of sampling and sample handling in strumentation, columns, and data handling. This paoper describes high precision computer measurements for characterizing capillary column efficiencies which are within 75% of the theoretical limit of capillary GC. Particular emphasis is given to detailed peak shape analysis, measurement accuracy and reproducibility, and system stability. Using known instrument performance parameters, it is then possible to characterize column performance with high accuracy in a meaningful manner. It is proposed that wall-coated tubular columns be characterized in terms of their chromatographic performance by the following parameters: Trennzahl (separation number), number of theoretical plates/meter, program temperature beseline stability, acid-base ratio, and the coefficient of skewness for 1-octanol. Statistical moments (m2) and hybrid moments are used to describe capillary column chromatographic performance because they may be related to basic physico-chemical column processes. These measurements are very sensitive parameters for characterizing GC columns. Using an online computer-based data system, the limits of capillary GC are shown to be limited by the sampling and injection steps.     

Capillary electrophoresis for thermodynamic and kinetic studies of peptidyl-proline isomerization by the theoretical plate height model

The theoretical plate height model, extended to include reactive CE, is used to calculate equilibrium constants and rate constants for the reversible, first-order isomerization of proline dipeptides. This model is consistent with chromatographic theory and enables calculation of equilibrium constants from velocity and calculation of rate constants from plate height. Thermodynamic and kinetic parameters for isomerization of Ala-Pro and Phe-Pro are calculated by using the plate height model, and are shown to be in good agreement with literature values. Additionally, the efficacy of the plate height model is compared to ChromWin, an existing simulation method for calculating rate constants from zone profiles. It is shown that ChromWin and the plate height model are complementary methods. ChromWin is best used for calculating rate constants for reactions that are far from steady state, where the zone profiles exhibit plateau formation. On the other hand, the plate height model is best used for calculating rate constants for reactions that are at or near steady state, where the zone profiles exhibit a single zone containing both reacting species.     

Development of an HPLC Assay Methodology for a Desonide Cream with Chemometrics Assisted Optimization

A new reversed-phase liquid chromatographic method for the separation and simultaneous quantification of desonide, sorbic acid, methylparaben, propyl gallate, and the major degradation product of desonide in a hydrophilic cream was developed with the aid of experimental design, resolution loss functions, and chemometrics methods. A strategy involving a screening phase and a fractional factorial design revealed the most influent chromatographic variables (pH and organic solvent content). The arc tangent resolution function was adopted as the optimization loss function. These variables were further optimized using a central composite design. Multivariate curve resolution and partial least squares regression were tested to optimize the chromatographic run time. The latter revealed to be superior in terms of precision and allowed the validation of a method with a total run time 3 times lower (approximately 8 min). The experimental design and chemometrics models enabled an efficient use of time and resources in predicting the optimum separation conditions for the desonide formulation. The validation of the resulting method according to the current ICH guidelines confirmed its selectivity, linearity, accuracy, and precision.     

Parameter selection for peak alignment in chromatographic sample profiling: objective quality indicators and use of control samples

In chromatographic profiling applications, peak alignment is often essential as most chromatographic systems exhibit small peak shifts over time. When using currently available alignment algorithms, there are several parameters that determine the outcome of the alignment process. Selecting the optimum set of parameters, however, is not straightforward, and the quality of an alignment result is at least partly determined by subjective decisions. Here, we demonstrate a new strategy to objectively determine the quality of an alignment result. This strategy makes use of a set of control samples that are analysed both spiked and non-spiked. With this set, not only the system and the method can be checked but also the quality of the peak alignment can be evaluated. The developed strategy was tested on a representative metabolomics data set using three software packages, namely Markerlynx™, MZmine and MetAlign. The results indicate that the method was able to assess and define the quality of an alignment process without any subjective interference of the analyst, making the method a valuable contribution to the data handling process of chromatography-based metabolomics data.     

药物分析中薄层色谱的方法认证

在药物分析中,针对所要求的性能参数,对一个薄层色谱程序的各个环节必须进行的认证方法和认可标准进行了讨论。建议当提出结果报告时,应附上关于对方法的认证参数和认证方法的说明。     

Statistical assessment of solvent mixture models used for separation of biological active compounds

Two mathematical models with seven and six parameters have been created for use as methods for identification of the optimum mobile phase in chromatographic separations. A series of chromatographic response functions were proposed and implemented in order to assess and validate the models. The assessment was performed on a set of androstane isomers. Pearson, Spearman, Kendall tau-a,b,c and Goodman-Kruskal correlation coefficients were used in order to identify and to quantify the link and its nature (quantitative, categorical, semi-quantitative, both quantitative and categorical) between experimental values and the values estimated by the mathematical models. The study revealed that the six parameter model is valid and reliable for five chromatographic response factors (retardation factor, retardation factor ordered ascending by the chromatographic peak, resolution of pairs of compound, resolution matrix of successive chromatographic peaks, and quality factor). Furthermore, the model could be used as an instrument in analysis of the quality of experimental data. The results obtained by applying the model with six parameters for deviations of rank sums suggest that the data of the experiment no. 8 are questionable.