Development Of High-performance Liquid Chromatography Method Research Articles

Initial experiments in high-performance liquid chromatographic method development I. Use of a starting gradient run

A single gradient elution run with acetonitrile (B)-water (A) as mobile phase can be used to estimate preferred conditions for subsequent method development experiments based on reversed-phase high-performance liquid chromatography. For a broad range of sample types,that includes both very hydrophilic and hydrophobic compounds, it was found that isocratic retention is given by log k ≈ log k w −4.2 ϕ, where ϕ = 0.01 %B. An initial gradient run allows values of log k w to be estimated for each compound in the sample, which then permits compound retention to be approximated as a function of either isocratic or gradient experimental conditions. The use of an initial gradient run in this way provides a rational basis for the subsequent development of a final HPLC method. Predictions based on this initial run can be used to (a) select between isocratic or gradient elution for further experiments and the final method, (b) choose a value of %B to achieve a desired value of k for the initial or final band in isocratic separation and/or (c) choose values of %B for the initial and final mobile phase in gradient elution. The present approach is based on a wide range of sample types and different reversed-phase columns; for this reason it is expected to be reasonably general and accurate. Errors in subsequent predictions based on an initial gradient run can also be estimated as a function of experimental conditions, allowing the selection of conditions for minimum error.

Initial experiments in high-performance liquid chromatographic method development II. Recommended approach and conditions for isocratic separation

Initial method development experiments for both neutral and ionic samples are best carried out with reversed-phase high-performance liquid chromatography (HPLC) using acetonitrile-methanol-buffer mobile phases. The preceding paper (Part I) suggests the use of an acetonitrile-buffer gradient to start method development. In this paper, optimum conditions for this first separation are discussed. Sequential method development experiments plus computer simulations are then used to obtain a final HPLC method. In this connection, we have examined how many experiments are required for reliable predictions of ternary-solvent retention. Three experiments are sufficient to predict isoeluotropic retention for methanol-acetonitrile-buffer ternaries where solvent strength does not vary, but five experiments are required for ternaries that contain tetrahydrofuran.

HPLC Method Development and Validation for Formaldehyde in Enteric Coating of Hard Gelatin Capsules

A reversed-phase high-performance liquid chromatography (HPLC) method for determination of formaldehyde in enteric coating of hard gelatin capsules is described and fully validated. This method entails the separation of formaldehyde as its 2,4-dinitrophenylhydrazone derivative using isocratic solvent eluition and its quantification with appropriate internal standard and ultraviolet detection. The results for selectivity, linearity, precision, accuracy and recovery were in agreement with validation parameters.

Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column.

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio- and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature suggested that reversed-phase HPLC with silica-based columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono- and dihydroperoxides as well as other unidentified products.

Effect of integration parameters on high-performance liquid chromatographic method development and validation

An HPLC method is defined as a specific setting of the physical, chemical and data processing parameters which control the chromatographic analysis. During the development or validation of a method no special attention is usually given to the data processing parameters. However, it turns out that HPLC methods for complex samples can be very sensitive to minor changes in certain numerical integration parameters such as the threshold. A series of statistically designed Chromatographic runs for a dyestuff with a very large number of peaks is presented where the interpretation of the data depends crucially on which threshold parameter value is chosen. It is therefore recommended that for development or validation of an HPLC method, especially in the context of quality control of complex substances, greater attention should be paid to the integration parameters.

Chiral separation by high performance liquid chromatography. I. Review on indirect separation of enantiomers as diastereomeric derivatives using ultraviolet, fluorescence and electrochemical detection.

The increased attention on the therapeutic implications of stereoisomerism has provided an impetus for the development of analytical methods for enantiomeric separation. The indirect method of separation of enantiomers as diastereomers using high performance liquid chromatography (HPLC) has emerged as an efficient and versatile approach. This is due mainly to the availability of numerous chiral derivatization reagents (CDRs). This article reviews CDRs useful for the development of an indirect HPLC method using ultraviolet, fluorescence and electrochemical detection. In addition, factors crucial for the development of the indirect method are discussed.

Use of solvent selectivity optimization procedures for high-performance liquid chromatographic method development

Solvent optimization procedures for high-performance liquid chromatography (HPLC) method development have been used in the past, but have never become routinely established owing to the complexity of the task involved. A unique solvent optimization software package has been developed by Philips Research that reduces the complexity of the problem. The optimization software utilizes four solvents and data from a UV diode-array system, and requires only eleven experiments in order to produce an optimized isocratic method. It has proved to be a powerful tool for solvent optimization in HPLC method development, and has proved its ability to resolve complex chromatographic problems, reducing method development time considerably. The system produces isocratic methods which, when compared with similar gradient methods, have analysis times reduced typically by a factor of 3-4. The fact that isocratic rather than gradient methods are produced leads to the production of more robust methods that can be easily transferred to other HPLC laboratories for routine testing or quality control purposes.

Determination of aminoglycoside antibiotics by reversed-phase ion-pair high-performance liquid chromatography coupled with pulsed amperometry and ion spray mass spectrometry

This work constitutes a preliminary investigation of a high-performance liquid chromatographic (HPLC)-mass spectrometric (MS) method for confirming aminoglycoside residues in bovine tissues. A reversed-phase ion-pair HPLC method for the separation of four aminoglycosides was developed using volatile ion-pairing agents and optimized for detection with an ion spray HPLC-MS interface. The method is also compatible with a commercial pulsed amperometric detector that was used for HPLC method development and that may be useful for the screening and quantification phases of a regulatory method. Several column phases, eluent compositions, and pairing ions were evaluated for optimum HPLC-MS sensitivity. Detection limits are in the low nanogram range with the pulsed amperometric detector and with HPLC-MS in the selected ion monitoring mode. Results with bovine kidney, fortified to 20 ppm and extracted by matrix solid-phase dispersion, obtained using both detectors are presented.

Integrating expert systems for high-performance liquid chromatographic method development

A complex expert system for high-performance liquid chromatographic method development has been obtained by linking several smaller stand-alone expert systems (modules), each representing a part of the entire domain. The modules LABEL, DASH and LIT select initial chromatographic conditions (first guess), the modules LABEL′, DASH′ and LIT′ perform retention optimization and the module SLOPES, which consists of the stand-alone expert systems VARIABLES, DESIRE and CRISE, is used for selectivity optimization. The linking is achieved through a supervisor, that is able to decide which of the former stand-alone expert systems must be activated at each stage of the development. The stand-alone expert systems were implemented by the use of the expert system building tool (ESBT) KES. To allow communication between the modules, they were embedded in C-language programs, without modification of the knowledge of the smaller systems.

Expert systems in chemical analysis

The expert systems in chemical analysis (ESCA) project was set up to evaluate the merits of expert system technology for use in industrial chemical analyses. Its aim is to provide expert systems that will illustrate the benefits and shortcomings of expert system technology. The area of interest is high-performance liquid chromatography (HPLC) method development in pharmaceutical analysis. In the first stage a number of tools were tested with respect to their suitability for expert system building in HPLC method development. Expert systems were then built for specific subdomains of HPLC method development. The subdomains together cover the entire method development process. In the final stage, some of the systems will be integrated to form larger systems that provide strategies for consistent and efficient method development.

Application of expert systems programming to high-performance liquid chromatography method development

Expert system programming methods are applied to problems in high-performance liquid chromatography (HPLC) method development, resulting in programs which can aid chromatographers. Knowledge related to HPLC is organized in a hierarchical structure with rules and facts at the base, followed by procedures, and ending with contexts for sets of rules. A final level of organization is division of major method development tasks into separate expert systems.

Expert system program for assistance in high-performance liquid chromatographic method development

A series of rule-based expert system programs which advise chromatographers during high-performance liquid chromatographic method development is described. The system is based on the VIS inference engine, and operates on both IBM-PC/AT compatible and VAX computers. The relationshiop of the programs to each other and to the chemist developing methods is described, and a coordinated example of program use is presented.

Practical approach for high-performance liquid chromatographic method development: assaying synthetic intermediates of a leukotriene inhibitor

High-performance liquid chromatographic (HPLC) methods for analyzing new drugs and their synthetic intermediates are needed as the synthesis is optimized and scaled up from making milligram amounts for initial evaluation of biological activity to producing kilogram amounts of the drug for thorough testing purposes. The most efficient solution is a single HPLC method that can be used for each step of the synthesis. A practical approach for the development of a single HPLC method is the use of computer-assisted method development to maximize the resolution within a reasonable analysis time. The computer program DryLab I was used in the development of an HPLC assay for the synthetic intermediates of a leukotriene inhibitor. The use of DryLab I with binary mixtures of organic solvents in the organic portion of reversed-phase HPLC systems is reported. With the retention data from two initial analyses, resolution can be optimized as a function of solvent strength.

Computer-assisted optimization in high-performance liquid chromatographic method development

Computer-assisted optimization in high-performance liquid chromatography has been encouraged by a need to define method development strategies for automated chromatographic instruments. Several algorthms have been developed for the optimization of various aspects of chromatographic performance. Additional experimental designs have been used to understand the influence of factors on the performance of the chromatographic system.

An evaluation of expert system tools on their use in high-performance liquid chromatography

Van Leeuwen, J.A., Vandeginste, B.G.M., Postma, G.J. and Kateman, G., 1989. An evaluation of expert system tools on their use in high-performance liquid chromatography. Chemometrics and Intelligent Laboratory Systems, 6: 239–252. The selection of a suitable tool for expert system development is often an important step in building an expert system. An evaluation of candidate tools is necessary before such a decision can be made. In this case four tools are evaluated for the implementation of an expert system on high-performance liquid chromatography method development. The evaluation is made on the basis of an implementation of a small test knowledge base in each of them. The test knowledge base is a part of a larger expert system that is under development. The decision in favour of a tool is based on an evaluation of the performance of the tool in relation to a number of evaluation criteria.

Sequential simplex optimization and multichannel detection in HPLC: Application to method development

The sequential simplex procedure for the optimisation of mobile phase composition in high-performance liquid chromatography (HPLC) is combined with multichannel detection. The operating software already available in a commercial LC system is exploited and extended to incorporate a number of new routines developed to facilitate HPLC method development. An efficient stop criterion for automated simplex procedures is proposed, based on continuous comparison of the chromatographic response function attained with that predicted. The theoretical basis is developed for a new peak homogeneity test, based on the wavelength sensitivity of the chromatographic peak maximum. An algorithm for assigning peak elution order, based on peak areas at multiple wavelengths, is developed for cases where multiple optima are recorded. The simplicity and practical utility of these techniques are demonstrated in the context of an HPLC separation method developed for a model system of six solutes.

High-performance liquid chromatographic method-development using computer simulation

Abstract Computer-assisted method-development schemes for high-performance liquid chromatography have taken another step forward with the introduction of 'computer simulation'. Software for the application of this technique is described and illustrated.

Band-spacing in reversed-phase high-performance liquid chromatography as a function of solvent strength: A simple and fast alternative to solvent optimization for method development

Numerous reports have described the use of solvent optimization for isocratic reversed-phase high-performance liquid chromatography method development. Solvent optimization involves the use of different solvents (usually methanol, acetonitrile and tetrahydrofuran) to control band-spacing for maximum resolution of the sample. Here, we examine an alternative approach, based on variation of the concentration of organic solvent in the mobile phase (solvent strength). This procedure is less powerful than classical solvent optimization, but it nevertheless possesses a significant ability to effect changes in band-spacing. It is also much more easily carried out. Many samples do not require solvent optimization, and in these cases, a change in solvent strength may be the more practical approach. The retention data required for solvent-strength optimization are most conveniently collected by using two initial gradient runs. The application of gradient retention data for developing a final isocratic separation is facilitated by the use of commercial software. The advantages and limitations of gradient-retention data for this purpose are examined.

Simultaneous determination of gatifloxacin and ambroxol hydrochloride from tablet dosage form using reversed-phase high performance liquid chromatography

A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm x 4.6 mm, 5 microm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70 : 30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10 -60 microg/mL and that for AM was found to be 5 - 30 microg/mL. The correlation coefficients were 0.999 6 and 0.999 3 for GA and AM respectively. The results of the tablet analysis (n = 5) were found to be 99.94% with +/- 0.25% standard deviation (SD) and 99.98% with +/- 0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92% - 100.02% and that of AM was 99.86% - 100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.

27. Allergic reaction to ‘bitter lemon’

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.