Reference compounds quantification

In Raman spectroscopy the intensity of scattered radiation depends not only on the polarizability and concentration of the analyte molecules, but also on the optical properties of the sample and the adjustment of the instrument. Absolute Raman intensities are not, therefore, inherently a very accurate measure of concentration. These intensities are, of course, useful for quantification under well-defined experimental conditions and for well characterized samples otherwise relative intensities should be used instead. Raman bands of the major component, the solvent, or another component of known concentration can be used as internal standards. For isotropic phases, intensity ratios of Raman bands of the analyte and the reference compound depend linearly on the concentration ratio over a wide concentration range and are, therefore, very well-suited for quantification. Changes of temperature and the refractive index of the sample can, however, influence Raman intensities, and the band positions can be shifted by different solvation at higher concentrations or... 

With respect to quantification of SPC in environmental samples, one has to deal with the scarcity of available reference compounds. Of the four SPC homologues, C6, C8, Cio and Cn, analysed in coastal waters after SPE [20], the response factor of C8-SPC in the ESI—MS was found to be about three times lower than those of the longer chain species. This was attributed to the much higher water content in the eluent at the time of elution of C8-SPC, which resulted in a notable reduction of the ionisation efficiency. This fact was taken into account [17] by quantifying individual SPC based on the responses of the two... 

REFERENCE COMPOUNDS IN QUANTIFICATION OF SURFACTANTS, THEIR METABOLITES AND REACTION BY-PRODUCTS... 

Source of contamination Compound Extraction Clean-up/treatment Identification/quantification Reference... 

These steroids give two peaks, both of which are quantified, the retention times are of the first peak. 16a-OH-Et is not an available reference compound so quantification is against the 16a-OH-An calibrant bThis steroid is quantified against the 7DHPT calibrant... 

For trace analysis, it is preferable to use a method that relies on the relative response factor for a compound against a reference compound. The areas of the compounds to be quantified are compared to the area of a reference compound, called an internal standard, present at a given concentration in each one of the samples (see Fig. 4.13). This approach can compensate for imprecision due to the injected volume and instrument instability between successive injections. It is superior to the preceding method where all of these factors influence the quantification. 

For the quantification of the identified compounds, genuine reference compounds are needed, which are often not available for degradation products and metabolites. 

Identification and quantification Two spectral user-libraries (MS and MS/MS) were developed using injections of TMS-derivatized authentic reference compounds. The MS library recorded the retention times and normal El mass spectra of trimethylsilyl (TMS) derivatives of authentic standards under the chosen chromatographic conditions, while the MS/MS library recorded retention times and product ion spectra derived from the specifically chosen precursor ions (Table 7.3) of TMS... 

In comparison with NMR, mass spectrometry is more sensitive and, thus, can be used for compounds of lower concentration. While it is easily possible to measure picomoles of compounds, detection limits at the attomole levels can be reached. Mass spectrometry also has the ability to identify compounds through elucidation of their chemical structure by MS/MS and determination of their exact masses. This is true at least for compounds below 500 Da, the limit at which very high-resolution mass spectrometry can unambiguously determine the elemental composition. In 2005, this could only be done by FTICR. Orbitrap appears to be a good alternative, with a more limited mass range but a better signal-to-noise ratio. Furthermore, mass spectrometry allows relative concentration determinations to be made between samples with a dynamic range of about 10000. Absolute quantification is also possible but needs reference compounds to be used. It should be mentioned that if mass spectrometry is an important technique for metabolome analysis, another key tool is specific software to manipulate, summarize and analyse the complex multivariant data obtained. 

The problem of the reference compound is inherent to most chemical concepts. By definition a suitable reference compound is a compound that possesses the same properties as the target compound with the exception of the electronic and structural features to be investigated. In most cases, such a compound cannot be found since changes in the (electronic) structure automatically lead to changes in all properties and hinder meaningful comparison. This is the reason why many chemical concepts are discussed at a qualitative rather than a quantitative level. In fact, as has been forcefully described by Binsch, attempts to quantify a concept very often lead to the collapse of the whole concept. This potential collapse-by-quantification problem exists for the concepts of homoconjugation and homoaromaticity just as it does for the concept of aromaticity. 

Quantitative analysis of the surface composition from the intensity of the peaks requires the quantification factors to be known. As with the peak position, it is possible to use reference compounds in order to determine these factors (taking care to correct for any surface segregation that might modify the observed composition compared to the expected composition). A series of carefully chosen binary compounds can be used to establish relative sensitivity factors for a large number of elements (Fig, 5.6). 

Furthermore, the chiral discrimination of monoterpenes has been recognized as one of the most important analytical techniques in flavor chemistry and pharmacology because the optically active stereoisomers have different sensory qualities and biological activities. HPLC offers powerful techniques for separation and quantification of enantiomers because of the progressive improvement of chiral chromatographic materials and chiral detectors such as optical rotatory dispersion (ORD) and circular dichroism (CD) detectors. In contrast, determination of chiral compounds by GC typically requires coinjection of the reference compound with known stereochemistry. An HPLC system equipped with a chiral detector, on the other hand, allows direct determination of the configuration of chiral compounds.84... 

Probably most investigations have used El ionization, but the greater sensitivity of Cl is in some instances attractive and may enable use of underiva-tized samples (de Witt et al. 1988) an application of negative-ion Cl for quantification is noted below together with other ionization procedures. Ideally, comparison should be made between the complete mass spectrum of the analyte and that of the reference compound. If very low amounts of the analyte are available, it may not be feasible to obtain a complete spectrum and it may be necessary to carry out the comparison by monitoring selected ions... 

Quantitative data were obtained by integration of selected ion chromatograms extracted from the total ion current. The ions used for quantification are presented in Table 1. For GC/MS-analyses of reference compounds the limit of quantitation (LOD) was 0.5 ng (signal to noise ratio 10 1). In Lippe river water samples concentrations of 10 ng/L resulted in similar signal to noise ratios due to matrix effects. Therefore these values were defined as limits of quantitation in the present study. 

The ions used for quantification and the signal accumulation time as well as the recoveries for the extraction and evaporating procedures are summarized in Tab. 2. An external four-point-calibration generated from a mixture of authentic reference compounds was used for quantification. The limit of quantitation (LOQ) was 0.01 pg/kg dry matter calculated from GC/MS-analyses of reference compounds (approx. 100 pg/pL of reference substances were determined with a signal to noise ratio of 10 1). 

Quantitative data of selected target compounds were obtained by integration of specific ion chromatograms extracted from the TIC. Injection volume and sample volume inaccuracies were corrected for by using internal standard compounds as a surrogate standard. An external four-point-calibration generated from reference compounds was used for quantification. 

If the ESR spectra of the paramagnetic entity in the imknown sample (M) and of the standard or reference compound (REF) have the same number of features spread over the same magnetic field range and if these spectra are integrated over the same scan range, then ESR allows the quantification of this paramagnetic entity according to Eq. 16 ... 

Tandem mass spectrometry (MS/MS) has attained an enviable status as an analytical tool to identify and quantify compounds in complex mixtures. MS/MS refers to the coupling of two stages of mass analysis, either in time or space. Of all the ionization techniques, only electron ionization (El) provides abundant sttuctural information. To obtain additional structure-specific information by other ionization techniques, it has become essential to perform MS/MS experiments [1,2]. MS/MS was first used in the late 1960s [3]. Since that time, its applications and popularity have continued to grow. Its major contributions are in the fields of structure elucidation of unknown compounds, identification of compounds in complex mixtures, elucidation of fragmentation pathways, and quantification of compounds in real-world samples. In recent times, several new generations of instruments have become available for tandem mass spectrometry applications. Basic concepts of tandem mass spectrometry and an account of these new developments are presented in this chapter. Additional reading material is listed at the end of the chapter. 

The method of choice for the determination of most vitamins is HPLC due to its high separation capability, its mild analytical conditions, and the possibility to use various specifically adapted detection methods, e.g., LTV, fluorescence, or MS detection. All fat-soluble vitamins and most water-soluble vitamins have chromophores suitable for UV detection. Separation of vitamers and stereoisomers can be achieved. If a higher sensitivity is required HPLC with fluorescence detection can be used, either directly (e.g., vitamins A and E) or after derivatization (e.g., thiamine). A further improvement in sensitivity and specificity has been achieved by introducing HPLC with mass spectrometric detection in vitamin analysis. Due to the structural information retrievable, e.g., molecular mass, fragmentation pattern, this is the method of choice for analysis of samples with complex mixtures or low vitamin concentrations. Examples for the use of HPLC-MS in vitamin analysis include the determination of 25-hydroxy-D3 and pantothenic acid. However, one drawback of mass spectrometry is the need for an isotopically labeled reference compound for reliable quantification. Due to the structural complexity of many vitamins, these reference compounds are often expensive and difficult to synthesize. An interesting unique application is the determination of vitamin B12 by HPLC-IPC-MS, which is possible due to its cobalt content. 

In this article we have introduced the Bruker SGF Profiling method for the authentication, verification and quality control of fiuit juices. In addition to tihe quantification of a large array of characteristic compounds, this fully automated NMR screening technique uses statistical models for the estimation of fhiit content or the origin of the juice. This analysis tool can show known and unknown deviations from normality. Currently, routines are under development to identify unknown deviations by constructing spectral patterns which can be compared to an existing reference compound database. ... 

Schug, K.A., Lindner, W. (2005) Stereoselective discrimination and quantification of arginine and N-blocked arginine enantiomers by formation and dissociation of calcium-mediated diastereomeric trimer complexes with a chiral reference compound using electrospray ionization-ion trap tandem mass spectrometry. 7. Am. Soc. Mass Spectrom., 16,825-834. 

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