Quantitative analysis validation procedures

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

There is a recent trend towards simultaneous CE separations of several classes of food additives. This has so far been applied to soft drinks and preserved fruits, but could also be used for other food products. An MEKC method was published (Lin et al., 2000) for simultaneous separation of intense sweeteners (dulcin, aspartame, saccharin and acesulfame K) and some preservatives (sorbic and benzoic acids, sodium dehydroacetate, methyl-, ethyl-, propyl- and isopropyl- p-hydroxybenzoates) in preserved fruits. Ion pair extraction and SPE cleanup were used prior to CE analysis. The average recovery of these various additives was 90% with good within-laboratory reproducibility of results. Another procedure was described by Frazier et al. (2000b) for separation of intense sweeteners, preservatives and colours as well as caffeine and caramel in soft drinks. Using the MEKC mode, separation was obtained in 15 min. The aqueous phase was 20 mM carbonate buffer at pH 9.5 and the micellar phase was 62 mM sodium dodecyl sulphate. A diode array detector was used for quantification in the range 190-600 nm, and limits of quantification of 0.01 mg/1 per analyte were reported. The authors observed that their procedure requires further validation for quantitative analysis. 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

Despite the widespread use of CE-MS for qualitative analysis, few quantitative applications have been pubhshed for routine analysis, and the vahdation of CE-MS methods according to generally accepted criteria is very uncommon. To our knowledge, only a few validation procedures are reported in the hterature. Although CE methods can be validated like chromatographic techniques, there are some specific characteristics to be discussed when quantitative determinations are expected. 

In 1977 Kolb and Pospisil proposed a method for the quantitative analysis of volatiles in solid samples [48] by using headspace extraction and gas chromatographic detection. The method, termed discontinuous gas extraction, is based on stepwise gas extraction, followed by a subsequent analysis of the extracted volatiles. The method theoretically calculates the total amount of analyte in a soUd sample after a few successive extractions and makes the quantitation of volatile analytes in soUd matrices possible. The proposed method was validated by measuring the styrene content in polystyrene by discontinuous gas extraction and by a procedure proposed by Rohrschneider in which the polystyrene is dissolved in dimethyl formamide (DMF) [49]. The two methods were in good agreement, which supported the validity of the discontinuous gas extraction. Kolb and Pospisil later elaborated the theoretical treatment of discontinuous gas extraction and in 1981 the method was re-named as multiple headspace extraction (MHE) [50]. 

On principle, pre- and post-column derivatization can be performed. Derivatization of complex samples prior to chromatographic separation is more problematic because matrix effects may alter reactions. Therefore, pre-column derivatization procedures are less suitable for quantitative analysis. Nevertheless quantitative procedures are described in the literature. Piretti et al. [71-72,247] analyzed peracetylated flavan-3-ol monomers and procyanidin dimers among other compounds in apple tissue after acetylation on a nitrile stationary phase under normal-phase conditions. Tarnai et al. [13] used the same approach in the analysis of procyanidins from cherry tissue. Incomplete acetylation was never observed [71], but so far validation data are not available. 

Like all classical quantitative analysis methods, NMR spectroscopy needs calibration, calibration standards and a validation procedure. The standard techniques are used for calibration external calibration, the standard addition method and the internal standard method. A fourth is a special NMR calibration method, the tube-in-tube technique. A small glass tube (capillary) containing a defined amount of standard is put into the normal, larger NMR tube filled with the sample for analysis. In most cases, there are slight differences in the chemical shift of corresponding signals of the same molecule in the inner... 

Because of the pervasive and pernicious occurrence of matrix effects, it is usually advisable to build a routine check on the extent of these effects into any method that has been shown to be subject to them, e.g. the ME/RE procedure (Matuszewski 2003) described in Section 5.3.6a. A particularly deceptive cause of ionization suppression that is not really a matrix effect is the mutual interference of an analyte and its co-eluting SIS (Liang 2003 Sojo 2003), discussed in some detail in Section 5.3.6a. While any level of suppression (or enhancement) of ionization efficiency is undesirable, the mutual suppression of analyte and an isotope-labeled SIS appear to be equal, with minimal effect on the validity of the quantitative analysis, although it may adversely affect limits of detection and quantification as a result of the... 

In particular, if complex reaction mixtures have to be analyzed quantitatively in real time, time-consuming calibration and validation procedures have to be considered. Such sophisticated methods might be mainly the choice in cases of quality and process control during production, but also for long-term in-depth analysis in process optimization studies. However, recent progress in chemometric analysis might lessen this drawback in the future Modern techniques such as multivariate curve resolution (MCR) promise quantitative determination without any calibration procedure in the near future [23, 24]. 

Sample Preparation Because large amounts of proteins are present in biological samples (except urine), conventional HPLC columns will not tolerate the direct introduction of these samples for quantitative analysis. Most bioanalytical assays have a sample preparation step to remove the bulk proteins from the samples [2], In addition, there are other important reasons for a sample preparation step when developing LC-MS/MS methods. These include the reduction of matrix components from the samples and minimization of ion suppression (also called matrix effects ) in the mass spectrometric detection [18]. Once a bioanalytical method has been developed, the method performance must remain consistent over the duration of the study. The results generated based on a validated method procedure should be free from systematic error and any other characterized errors and meet the predefined acceptance criteria. Sample preparation is used to... 

Another approach is to perform a cross-validation much like that for quantitative models. However, rather than predicting the constituent values as each sample is rotated out (and there are none to predict in discriminant analysis anyway), the Mahalanobis distance of each sample is predicted at every factor. The cross-validation procedure is basically the same remove a sample or set of samples, construct a Mahalanobis matrix for one factor, two factors, etc., and then predict the sample(s) left out against it. The samples are then returned to the training set, and a new set is removed. The process is continued until every sample has been rotated out once. 

One of the main purposes of measuring NIR data is the determination of chemical composition or physical properties in a quantitative way. The principle of the measurement procedure for quantitative analysis is based on recording the NIR spectra of reference samples (the number depending on the number of components or parameters to be determined) of known composition. The levels of the constituents or the physical parameters are determined by independent, conventional analytical or physical methods. Then the set of reference spectra and the independently determined values of the parameters under investigation are used by a selected statistical method to build a calibration. This enables unknown samples to be evaluated with regard to the individual parameters of interest. The accuracy of the NIR technique depends upon the validity of the calibration data set, which must incorporate the entire range of concentrations that will be determined by the instrument. This set must contain samples with varying ratios of each component. NIR calibrations do not typically extrapolate or interpolate well across concentrations. Typical calibration sets include more than... 

On the other hand, the molar fraction must he constant in the particular solution and this is the fact when Xj is in the interval 320-360 nm, that is, statistically meaningful result can be obtained when there is a plateau. The narrowing of the decomposition interval, as shown in Figure 2.3, leads to narrowing of this plateau or even to the impossibihty to obtain statistically valid results for molar fractions. The latter happens when the band decomposition procedure is apphed only in the spectral interval 340-600 nm. Therefore, when this algorithm for quantitative analysis of tautomeric mixtures is apphed, the band decomposition interval must be as wide as the available spectral instrument and the used solvent would allow. 

Particularly for direct microanalytical techniques using <10 mg of sample for analysis, it is highly desirable to obtain quantitative information on element- and compound-specific homogeneity in the certificates for validation and quality control of measurements. As the mean concentration in a CRM is clearly material-related, the standard deviation of this mean value should represent the element s distribution in this matrix rather than differences in the analytical procedures used. 

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