Liquid chromatographic methods

ATP Verification Studies and Establishment of the Method Operable Design Region 

The purpose of method validation is to demonstrate that an analytical method is suitable for its intended purpose and, for a quantative method, provides a reasonable estimate of the true value of the sample tested. Appropriate performance characteristics, such as accuracy and precision, must be demonstrated before making decisions based on test data. Method validation involves assessing method performance against predefined criteria, established based on the sample specifications and the type of measurement to be performed, for example, assay, identification, or limit test. A rigorous assessment of method performance versus predefined criteria provides assurance that the method will consistently provide a fit for purpose performance. Method characteristics to be evaluated during method validation are described by several guidelines [1,2] some of which are shown in Tables 3.1 and 3.2. 

The focus of this chapter is validation of liquid chromatographic methods. Numerous articles describe details and provide examples for LC method validation [6,7] therefore, a step-by-step guide to method validation is not provided here. This chapter provides an overview of LC method validation as well as a discussion about some of the pitfalls associated with traditional validation and some alternative practices that are currently being discussed in the literature. 

The common LC method validation characteristics are listed in Table 3.2. Also listed in Table 3.2 are definitions for the different validation characteristics according to the International Conference on Harmonization 

International Conference on Harmonization (ICH) Validation of Analytical Procedures [1] Guidance for analytical methods presented in pharmaceutical applications filed in Europe, USA, and Japan 

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Current interest is, however, mainly in the coupling of HPLC and TLC, to which considerable attention has been devoted for the solution of difficult separation problems. Since Boshoff et al. (39) first described the direct coupling of HPLC and TLC, several papers (40-43) have been published describing the on-line coupling of liquid chromatographic methods and PC, usually with different interfaces, depending on the first technique applied. If PC is used as the second method, all the MD methods discussed above can be applied to increase the separating power. 

It follows that measurements must be made with a precision of about 0.2 second if quantitative results are to be of any value. It is seen from figure 4 that the experimental points lie very close to the line and a fairly accurate measurement of the distribution of the two isotopes can be obtained from retention time measurements. This method has very limited areas of application and is given here, more to demonstrate the effect of unresolved impurities on retention time, than to suggest it as an alternative to adequate chromatographic resolution. In some cases, however, particularly in the analysis of isotopes, it may be the only practical way to obtain a quantitative evaluation of the mixture by a liquid chromatographic method. 

High-Performance Liquid Chromatographic Method Applied to Parafytic Shellfish Poisoning Research... 

MAiANi G, SERAFiNi M, SALUCCi M, AZZiNi E and FERRO-Luzzi A (1997) Application of a new high-performance liquid chromatographic method for measuring selected polyphenols in hiunan plasma , J of Chromatog B, 692, 311-17. 

Coffey, J.S. et al., A liquid chromatographic method for the estimation of Class HI caramel added to foods. Food Chem., 58, 259, 1997. 

Chen, J.P. Tai, C.Y., and Chen, B.H., Improved liquid chromatographic method for determination of carotenoids in Taiwanese mango (Mangifera indica L.), J. Chro-matogr. A, 1054, 261, 2004. 

Breithaupt, D.E. and Schwack, W., Determination of free and bound carotenoids in paprika Capsicum annuum L.) by LC/MS, Eur. Food Res. TechnoL, 211, 52, 2000. Epler, K.S., Ziegler, R.G., and Craft, N.E., Liquid chromatographic method for the determination of carotenoids, retinoids and tocopherols in human serum and in food, J. Chrvmatogr, 619, 37, 1993. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Snyder, L. R., Dolan, J. W. Initial experiments in high-performance liquid chromatographic method development. I. Use of a starting gradient run. J. Chromatogr. A 1996, 721, 3-14. 

Tokunaga R, Takahata S, Onoda M, Ishi-i T, Sato K (1974) Evaluation of the exposure to organic solvent mixture. Comparative studies on detection tube and gas-liquid chromatographic methods, personal and stationary sampling, and urinary metabolite determination. Internationales Archiv Arbeitsmedizin 33 257-267. 

Liquid chromatographic methods are well suited to the separation and determination of metal chelates that can be extracted into organic solvents. Many chelates also absorb strongly in the UV onAisible regions, facilitating detection. The... 

Swadesh, J. K., Stewart, Jr., C. W., and Uden, P. C., Comparison of liquid chromatographic methods for analysis of homologous n-alkyl esters of biphenyl-4,4,-dicarboxylic acid, Analyst, 118, 1123, 1993. 

Marvin, C. H., Brindle, I. D., Singh, R. P., Hall, C. D., and Chiba, M., Simultaneous determination of trace concentrations of benomyl, carbendazim (MBC) and nine other pesticides in water using an automated on-line pre-concentration high-performance liquid chromatographic method, /. Chromatogr., 518, 242, 1990. 

Henricsson, S. and Westerholm, R., Liquid chromatographic method for analysing the colour marker Solvent Yellow 124, N-ethyl-N-[2-(l-isobutoxy-ethoxy)ethyl](4-phenylazophenyl)amine, in diesel fuels, ]. Chromatogr. A, 723, 395, 1996. 

Virtanen, V. and Lajunen, L. H. J., High-performance liquid chromatographic method for simultaneous determination of clodronate and some clodronate esters, /. Chromatogr., 617, 291, 1993. 

Figure 4.10 Classification of liquid chromatographic methods depending on the polarity of the stationary and mobile phases. After Schaffer el at. [537]. Reproduced by permission of Metrohm AG, Herisau, Switzerland...

N. A. Parris, Instrumental Liquid Chromatography, a Practical Manual on High-Performance Liquid Chromatographic Methods (Journal of Chromatography Library, Vol. 27), Elsevier, Amsterdam, 2nd revised ed., 1984 J. Drozd, Chemical Derivatization in Gas Chromatography (Journal of Chromatography Library, Vol. 19), Elsevier, Amsterdam, 1981 J. F. Lawrence and... 

Kurttio, P, Vartianen, T., and Savolainen, K. (1988) High-performance liquid chromatographic method for the determination of ethylenthiourea in urine and on filters, Analytical Chemistry Acta, 212 297-301. 

E Ruckenstein, V Lesins. Classification of liquid chromatographic methods based on the interaction forces The niche of potential barrier chromatography. In A Mizrahi, ed. Advances in Biotechnological Processes, Vol 8 Downstream Processes Equipment and Techniques. New York Alan R. Liss, 1988, pp 241-314. 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Hutta, M., Rybar, I., Chalanyova, M. (2002). Liquid chromatographic method development for determination of fungicide enantiomers by achiral and chiral column switching technique in water and soil. J. Chromatogr. A 959, 143-152. 

Schleyer, E., Reinhardt, J., Unterhalt, M., Hiddemann, W. (1995). Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine. J. Chromatogr. B 669, 319-330. 

Soltes, L., Sebille, B. (1997). Reversible binding interactions between the tryptophan enantiomers and albumins of different animal species as determined by novel high performance liquid chromatographic methods an attempt to localize the d- and L-tryptophan binding sites on the human serum albumin polypeptide chain by using protein fragments. Chirality 9, 373-379. 

In this chapter, the main analytical techniques and the methods currently employed in industrial and research laboratories for the analysis of important classes of additives are reviewed. The use of both gas chromatographic and liquid chromatographic methods coupled with mass spectrometry features prominently. Such methodology enables the sensitive and specific detection of many types of organic additives in polymeric materials to parts per billion (jig/kg) levels. Much of the development of these methods has been undertaken as part of research into the migration or extraction of species from food-contact and medical materials [5-7], This chapter also includes some discussion on the analysis of residual monomers and solvents. 

Tab. 2.3. Liquid-liquid chromatographic methods for the measurement of log D. Selection of column and mobile phase.

Tab. 2.4. Liquid-liquid chromatographic methods for measurement of log D at pH 7.4. Results for a series of validation compounds.

Erk and Altun [24] used a ratio spectra derivative spectrophotometric method and a high performance liquid chromatographic method for the analysis of miconazole nitrate and metronidazole in ovules. The spectral method depends on ratio spectra first derivative spectrophotometry, by utilizing the linear relationship between substances concentration and ratio spectra first derivative peak amplitude. The ratio... 

Sternson et al. [58] used a high performance liquid chromatographic method for the analysis of miconazole in plasma. Miconazole was extracted from alkalinized plasma with n-heptane-isamyl alcohol (98.5 1.5) and separated by high performance performance liquid chromatography on p-Bondapak Ci8 with ultraviolet detection at 254 nm. The mobile phase was methanol-tetrahydrofuran-acetate buffer (pH 5) (62.5 5 32.5) containing 5 mmol octanesulfonate per liter. The flow rate was 2 mL/min. Recovery was 100%. The relative standard deviation for injection-to-injection reproducibility was 0.4% and that for sample-to-sample variation was 5% at high miconazole concentrations (30 pg/mL) and 1% at low (1 pg/mL) concentrations. The limit of detection was 250 ng/mL. 

Fan used a high performance liquid chromatographic method for the qualitative and quantitative analysis of miconazole [59], Miconazole sample was dissolved in methanol and determined by high performance liquid chromatography using methanol-water (75 25) as the mobile phase and ultraviolet detection at 214 nm, the recovery was more than 99.4% and the accuracy was satisfactory for the qualitative and quantitative analysis. 

Guillaume et al. [69] presented a high performance liquid chromatographic method for an association study of miconazole and other imidazole derivatives in surfactant micellar using a hydrophilic reagent, Montanox DF 80. The thermodynamic results obtained showed that imidazole association in the surfactant micelles was effective over a concentration of surfactant equal to 0.4 pM. In addition, an enthalpy-entropy compensation study revealed that the type of interaction between the solute and the RP-18 stationary phase was independent of the molecular structure. The thermodynamic variations observed were considered the result of equilibrium displacement between the solute and free ethanol (respectively free surfactant) and its clusters (respective to micelles) created in the mobile phase. 

Gagliardi et al. [72] developed a simple high performance liquid chromatographic method for the determination of miconazole and other antimycotics in cosmetic antidandruff formulations. This high performance liquid chromatographic method was carried out on a Discovery RP Amide Ci6 column and spectrophotometric detection was performed at 220 nm. The initial mobile phase was a mixture of acetonitrile and aqueous 0.001 M sodium perchlorate (pH 3) in the ratio of 15 85 (v/ v) then a linear gradient less than 46% acetonitrile in 70 min, and less than 50% in 80 min. The extraction procedure was validated by analyzing samples of shampoo... 

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