HPLC methods developing equivalent

The selectivity of reversed-phase liquid chromatography (RP-LC) columns is known to vary, even columns with the same ligand (e.g., Cjg). Column selectivity can also vary from batch to batch for columns claimed to be equivalent by the manufacturer. For different reasons, it is sometimes necessary to locate a replacement column for a given assay that will provide the same separation as the previous column. In other cases, as in HPLC method development, a column of very different selectivity may be needed - in order to separate peaks that overlap on the original column. For each of these situations, means for measuring and comparing column selectivity are required. Until recently, no such characterization of column selectivity was able to guarantee that two different columns can provide equivalent separation for any sample or separation conditions. 

The near-IR technique has been used very successfully for moisture determination, whole tablet assay, and blending validation [23]. These methods are typically easy to develop and validate, and far easier to run than more traditional assay methods. Using the overtone and combination bands of water, it was possible to develop near-IR methods whose accuracy was equivalent to that obtained using Karl-Fischer titration. The distinction among tablets of differing potencies could be performed very easily and, unlike HPLC methods, did not require destruction of the analyte materials to obtain a result. 

Method development can be made easier, quicker, and more effective by the use of computer simulation. DryLab, which has been in use in hundreds of laboratories around the world during the past 20 years, has been developed to the point where the user can simulate any combination of any two experimental conditions that affect separation selectivity. At the same time, this software also allows the user to study the effects of changing column dimensions, particle size or flow rate, in either an isocratic or gradient mode. More recently, DryLab has been incorporated into HPLC systems which allow unattended method development (Waters Corp., Milford, MA). In all of these applications, predictions by DryLab have been shown to be essentially equivalent to actual experiments - while greatly reducing the cost and time required in method development. 

The method developed using the Poroshell column was equivalent to the HPLC method, with a similar gradient slope. Due its higher linear velocity, the run time was decreased twofold. 

In TLC the detection process is static (sepaurations achieved in space rather than time) and free from time constraints, or from interference by the mobile phase, which is removed between the development and detection process. Freedom from time constraints permits the utilization of any variety of techniques to enhance detection sensitivity, which if the methods are nondestructive, nay be applied sequentially. Thus, the detection process in TLC is nore flexible and variable than for HPLC. For optical detection the minimum detectable quantities are similar for both technlqpies with, perhaps, a slight advantage for HPLC. Direct comparisons are difficult because of the differences in detection variables and how these are optimized. Detection in TLC, however, is generally limited to optical detection without the equivalent of refractive... 

A gas chromatographic (GC) method has been described in the literature. GC is based on the oxidation of microcystins which splits the Adda side chain to produce 3-methoxy-2-metlyl-4-pheitylbu-tyric acid (MMPB), which is then determined, either by GC or GC/MS (as its methyl ester) (Sano 1992 Kaya and Sano 1999) or by HPLC/fluoiescence detection (after conversion to a fluorescent derivative) (Sano 1992). GC/MS has been used to monitor microcystins in Japanese lakes (Tanaka 1993) and in sediments (Tsuji 2001). A similar method was developed by Harada (1996), but in this case the MMPB was determined directly without derivatization using GC/MS or LC/MS. The results of this approach ate given in terms of total toxin concentration, which then can be expressed in terms of microcystin-LR. However, individual toxins ate not determined and consequently it is not possible to produce a result in terms of microcystin-LR toxicity equivalents. This procedure cannot therefore be used to monitor water samples in relation to the proposed guideline. 

The ultimate development in the field of sample preparation is to eliminate it completely, that is, to make a chemical measurement directly without any sample pretreatment. This has been achieved with the application of chemometric near-infrared methods to direct analysis of pharmaceutical tablets and other pharmaceutical solids (74-77). Chemometrics is the use of mathematical and statistical correlation techniques to process instrumental data. Using these techniques, relatively raw analytical data can be converted to specific quantitative information. These methods have been most often used to treat near-infrared (NIR) data, but they can be applied to any instrumental measurement. Multiple linear regression or principal-component analysis is applied to direct absorbance spectra or to the mathematical derivatives of the spectra to define a calibration curve. These methods are considered secondary methods and must be calibrated using data from a primary method such as HPLC, and the calibration material must be manufactured using an equivalent process to the subject test material. However, once the calibration is done, it does not need to be repeated before each analysis. 

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