Two-step quantification

Use the peak contours in multidimensional space that facilitates refinements in quantitation through two-step quantification approach (see Chapter 15) in combination with corrections for mass offset and isotopologue analysis in a bioinformatics manner. 

Figure 14.2 Two-step quantification of sphingomyelin and galactosylceramide species from lipid samples of mouse cortices. Lipid samples were prepared after treatment of mouse cortex lipid extracts with lithium methoxide as previously described [38]. MDMS-SL analysis of lipid samples was conducted in the positive-ion mode after addition of a small amount of LiOH. The MS trace a was the mass spectrum of the lipid samples acquired in the full MS mode. The MS trace b displayed a mass spectrum of NLS183, corresponding to phosphocholine, characteristic of sphingomyelin (SM) species in the solution under the conditions. The MS trace c showed a mass spectrum of NLS162, corresponding to a hexose derivative that represents a characteristic building block of HexCer species in the solution under the experimental conditions. IS denotes internal standard. Each spectrum displayed is normalized to the base peak in the spectrum.

Two caveats of this two-step quantification approach should be recognized. 

This two-step quantification approach cannot be applied to any lipid class for which a class-specific and sensitive PIS or NLS is not present, e.g., TAG, Cer, PE, and CL. Special quantification approaches for these lipid classes have been developed in MDMS-SL with or without derivatization [42 5],... 

Quantification of rate constants for this multistep process hinges on the assumed rate-controlling step. Depending on the steps that are assumed to dictate the rate, reaction rates or diffusion constants are calculated from the net kinetics of reaction or sorption. Various studies have assumed that either of two steps are rate controlling either the surface diffusion or the actual spillover from the source. All analyses have assumed a first-order dependence of the concentrations of atomic hydrogen for each step in the sequence. 

The principle of the toxicity classification system for wastes discharged into the aquatic environment is a two-step determination and quantification of the acute toxicity of the liquid wastes or leachates with the battery of bioassays. In the first step the toxicity is determined on non-diluted samples, and in the second step the toxicity tests are performed on a dilution series of the samples with all bioassays for which more than 50% effect has been found in the non-diluted sample. 

SPME consists of two steps extraction and desorption. In the extraction process the fiber is immersed into the sample with a syringe, vigorous stirring is applied, and the organic micropollutants are retained in the fiber depending on their distribution coefficients. Then, using the holder, the fiber is transferred to the analytical instrument for desorption, separation and quantification. The method has been automated and commercial systems are available which will extract, agitate, and inject the sample into a GC system. In HPLC the sample is extracted directly into the eluent stream rather than thermally desorbed." ... 

SPME was hrst used by Pawliszyn et al. in 1990. It is a two-step process conductive to the simultaneous extraction and preconcentration of analytes form sample matrices. In the first step, a fused-silica fiber coated with a polymeric stationary phase is exposed to the sample matrix where the analyte partitions between the matrix and the polymeric stationary phase. In the second step, the fiber/analyte is transferred to the analytical instrument for desorption, separation, and quantification. SPME has a number of advantages over traditional extraction techniques for pesticides. In fact, it is fast, simple, solvent-free, and easily automated for both GC and HPLC instruments. It exhibits good linearity and sensitivity. Thus, carbamate and organophosphorus pesticides in golf course samples were successfully extracted by SPME and analyzed by HPLC by Jinno et al. ... 

Lerke et al (1983) developed a rapid screening method to detect histamine in fish. The qualitative procedure uses a two-step sequential enzyme system. In the first step, the enzyme diamine oxidase catalyzes the breakdown of histamine with production of hydrogen peroxide. Detection of hydrogen peroxide is then performed by the formation of crystal violet from the leuco base in the presence of peroxidase at 596 nm. The method could be used to detect histamine in raw or heat-processed fish. Later, Lopez-Sabater et al (1993) modified the procedure to achieve histamine quantification. Further modification of the Lerke method by Rodriguez-... 

The process to convert experimental XRF data into analytically useful information (usually in the form of concentration values of elemental constituents whose X-ray peaks are visible above the background in the spectrum) can be divided into two steps first the evaluation of the spectral data, whereby the net height or the net intensity of the X-ray peaks is determined, taking care to correct for peak overlap (if any) between X-ray lines of different elements and secondly the conversion of the net X-ray intensities into concentration data, i. e. the quantification. In this last step, especially, the appropriate correction of matrix effects is a critical issue. 

A two-step approach was used for the determination of method detection and quantification limits for the sulfur analytes, as described in Lee Aizawa (2003). The two step approach takes into consideration several factors that affect the analyte signal, including instrumental noise, variability in instrumental sensitivity, and variability in method efficiency, matrix effects and interference, and is simple to follow. Other methods, such as the Hubaux-Vos approach for the calculation of the detection limit can also be used, as reported in Fedrizzi et al. (2007). However, this later approach is complicated, time consuming and does not take either the variabiUty in method efficiency or the matrix effects into consideration (Lee Aizawa, 2003). A brief discussion on how to conduct the method vahdation using the two steps approach is mentioned in this section. 

The approach described in Lee Aizawa (2003) consists of two steps for the determination of the LOD and LOQ. These firstly involve determination of the Instrumental Detection Limit (IDL) and Instrumental Quantification Limit (IQL), and using these values to estimate the Method Detection Limit (MDL) and Method Quantification Limit (MQL), following calculation of the LOD and LOQ for the extraction/analysis method. 

Fig. 17 Cyclic, thermomechanical tensile test for quantification of triple-shape effect - two step programming, (a) Strain and temperature as a function of time taken from the fifth cycle for MACL(45) multiphase network composed of crystallizable PCL segments and amorphous poly(cyclohexyl methacrylate) segments with 45 wt% PCL content (rtrans,A = Tm.PCL = 50°C and Ftrans.B = = Fg pcHMA = 140°C). The solid line indicates strain the dashed line indicates tem-...

After separation of different lipid classes in the ESI source (i.e., intrasource separation) and MDMS identification of individual species (see Section 3.2.3.3), quantification of the identified individual species of a lipid class of interest is performed in a two-step procedure in MDMS-SL [11, 35]. This procedure can be conducted automatically [29]. 

A normalization parameter (e.g., cell dry weight or protein content) should be selected and determined [16] so that final results can be compared between the samples. It is advised that at least one internal standard of each lipid class and subclass of interest should be added to the cell lysates prior to lipid extraction for quantification, profiling, or relative comparison regardless of which approach is employed for lipid analysis. Lipids from the harvested yeasts are commonly extracted utilizing solvents. Different solvent systems were used for the purpose (e.g., Bligh-Dyer extraction [18], two-step extraction [19], and ethanol-based extraction [16]). It appears that all of the extraction methods deseribed in Chapter 13 could be used. 

This two-step decomposition procedure offers areasonable balance between avoiding bias in curve fitting, insuring the reproducibility required for a comparison of samples and providing a consistent basis for quantification. Whatever the procedure, it should be clearly stated when quantitative data are extracted from peak decomposition. 

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