External standards, spectrophotometric

Quantitative data can be obtained by means of the use of an internal or external standard, whose exact concentration can be checked by means of spectrophotometric detector, which measures spe-cihc absorbance or molar absorbance. Spiked authentic samples can be used to measure the limit of detection (LOD) and the limit of quantihcation (LOQ). 

An accurately prepared standard solution of retinyl acetate (i.e., a solution of known concentration) is taken through the saponification and extraction procedure along with each batch of samples, and the resultant retinol solution is used as an external standard without spectrophotometric standardization. This technique, which is recommended by COST 91 (149), compensates for losses of vitamin A incurred during the saponification and subsequent manipulations (i.e., the calculated vitamin A value is recovery-corrected). 

Table 6.1 displays recommended specifications for wavelength accuracy, photometric linearity, and spectrophotometric noise levels for pharmaceutical applications. The first step in validating any NIR method is to test the suitability of these specifications for a given application. Wavelength accuracy tests conducted using appropriate external standards will prevent potential problems that could occur with proprietary internal calibration protocols. The exact nature of any calibration standard must be noted in a validation protocol. Rare earth oxides and glass standards are candidates for such calibration. 

Since, In practice, the efficiency frequently varies, it is necesseuy to determine the counting efficiency before one can compare different samples. A radioactive standard of acciirately known activity is essential for the determination of the efficiency counting. The use of radioactive standards is in principle similar to that of standards in colorimetric or spectrophotometric assays. There are two types of standards (i) internal standard (this is usually a p-emitter of accurately known activity which, when dissolved in unquenched scintillation mixture, provides a reference standard), and (ii) external standard (ay-emitter incorporated in most instruments). 

It should be noted that Ellman s assay does not require the use of external standards, or the generation of a standard curve, which is very convenient. However, it is limited in its sensitivity. These assay limitations are directly because of the inherent limitations in spectrophotometric detection. For instance, if an Ellman s assay sample shows an absorbance of 0.05 at 412 nm (approaching the lower limit of detection), the thiol concentration is calculated to be 3.7 /zM. This may be suitable when assaying proteins in the mg/mL concentration range (1 mg/ mL bovine serum albumin =15 uM), but not for lower concentrations. In some cases, the presence of unmodified cysteine thiols is aberrant or undesirable a more sensitive assay to detect low levels of such species would be useful. 

After two decades of quantitative l.c. analyses, it has been established that, when proper precautions are taken, these methods can provide accurate and reproducible results.Quantitative l.c. measurements are usually as accurate as, and often more precise than, those obtained by spectrophotometric, " paper-chromatographic, and gas-liquid-chro-matographic methods. Both external and internal standardization have been used to translate peak height or areas into quantitative, solute-concentration values. Because peak heights are easy to measure, many methods use this parameter, and, when slightly overlapping peaks or unsteady baselines are encountered, it is the method of choice. With introduction of... 

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