Quantification stable isotope standards

With BBSs, the internal standard is present in the extraction solvent and can only equilibrate with the endogenous metabolites that are extracted from the DBS. Therefore, isotope dilution principles do not apply to the extraction step since they are not in the blood (blood spot) to begin with. However, subsequent steps such as derivatization, sample transfer, and so on do indeed follow the principles of isotope dilution. To note the differences between analyzing liquid versus BBSs using stable isotope standards and the fact that quantification is less accurate using BBSs, the term pseudoisotope dilution is often used. 

As described in 2.2.3.1, Principles of Assay , tHcy must be produced by chemical reduction, which is achieved in the method described here by dithiothreitol. tHcy is analysed by HPLC separation followed by electrospray ionisation and then separation of the ionised molecule in the first mass spectrometer, then fragmentation into a specific ion fragment in the second. Quantification is based on comparison of the signal from natural Hey (transition m/z 135.9 —< m/z 89.9) with that of the stable isotope internal standard (transition m/z 139.9 —< m/z 93.9). 

AdoMet and AdoHcy are separated by HPLC and analysed by electrospray ionisation-tandem mass spectrometry. Quantification is based on comparison of the signal from natural AdoMet (transition m/z 399 ->- m/z 250) and AdoHcy (transition m/z 385 ->- m/z 135 and 134) with that from analogous transitions of the stable isotope internal standards. 

Perhaps no reagent is more important than the stable-isotope-labeled internal standard in any clinical assay utilizing mass spectrometry for quantification. Internal standards are important in many aspects of the analysis and are somewhat different than standards utilized in other clinical, non-mass-spectrometric assays. The ideal internal standard is an enriched isotopic version of the analyte being measure. For example, in the case of phenylalanine, a standard available may contain six 13C molecules rather than 12C in the aromatic ring. This has the net effect of shifting the mass of phenylalanine by six units while also maintaining nearly identical chemical... 

Lanckmans, K., Sane, S., Smolders, I., and Michotte, Y. (2007). Use of a structural analogue versus a stable isotope labeled internal standard for the quantification of angiotensin IV in rat brain dialysates using nano-liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21 1187-1195. 

Martens-Lobenhoffer et al. [119] used chiral HPLC-atmospheric pressure photoionization tandem mass-spectrometric method for the enantio-selective quantification of omeprazole and its main metabolites in human serum. The method features solid-phase separation, normal phase chiral HPLC separation, and atmospheric pressure photoionization tandem mass spectrometry. The internal standards serve stable isotope labeled omeprazole and 5-hydroxy omeprazole. The HPLC part consists of Agilent 1100 system comprising a binary pump, an autosampler, a thermo-stated column component, and a diode array UV-VIS detector. The enantioselective chromatographic separation took place on a ReproSil Chiral-CA 5 ym 25 cm x 2 mm column, protected by a security guard system, equipped with a 4 mm x 2-mm silica filter insert. The analytes were detected by a Thermo Scientific TSQ Discovery Max triple quadrupole mass spectrometer, equipped with an APPI ion source with a... 

Stable-isotope-labeled compounds are the most important standard reagents used in most MS-based assays that require accurate quantification. Measurement of metabolites using stable isotopes is commonly referred to as isotope... 

The quantification of metabolites in dried blood spots primarily ensures that the quality of the isotopes standards is excellent in terms of chemical and isotopic purity. When using MS/MS, it is essential that the fragments produced by the collision cell and the product ions detected ensure that both labeled and unlabeled metabolites are identical. Most importantly, the choice of the isotope label and the structural positions must be such that they are stable and do not exchange with other isotopes during sample preparation. Finally, it is imperative that the mass shift is sufficiently high (at least 3 Da) for small molecules less than 1000 Da and that the label occurs at a mass free from other compound interference. Figure 4 illustrates the concepts of quantification using stable isotope with Phe measurement in a dried blood spot as an example. 

FIGURE 4 (A) The use of stable isotope internal standards for quantification of Phe extracted... 

Gas chromatography-selected ion monitoring (GC-SIM) is often used for the quantification of GAs by GC-MS, an internal standard labeled with stable isotopes (usually with deuterium) being used as the most reliable and sensitive method. A mass chromatogram reconstructed from full-scan GC-MS is also used for semiquantification. The amounts of GAs are determined by measuring the peak areas of ions characteristic for each GA and comparing these areas to those of authentic samples. When an internal standard labeled with a stable isotope is used, the ratio of the area of an ion peak characteristic for a sample and the area of the corresponding peak of the labeled internal standard is used for quantification. The analysis of GAs by GC-MS has been discussed in numerous publications and review articles.254-256... 

Quantification of aroma compounds using GC and internal reference has long been a problematic issue.81 Detection response factor, peak shape, discrimination phenomenon at the injector port, and, of course, disproportion during sample preparation were more or less unavoidable. Stable isotopes used as internal standards combined with an MS detector have realized reproducible and far more accurate quantification. The major drawback of this method is the tedious process of preparing the isotope-labeled standards. 

A compound or material that is not an analyte but is included in an unknown or standard to correct for issues in the processing or analysis of an analyte or analytes an internal standard is not a calibration standard. See Julka, S. and Regnier, F, Quantification in proteomics through stable isotope coding a review, J. Proteome Res. 3, 350-363, 2(X)4 Bronstrup, M., Absolute quantification strategies in proteomics based on mass spectrometry. Expert Rev. Proteomics 1, 503-512, 2004 Coleman, D. and Vanatta, L., Statistics in analytical chemistry, part 19-intemal standards, American Laboratory, December 2005. 

Several methods have been developed to quantify the F2-IS0PS. Our laboratory uses a gas chromatographic/negative ion chemical ionization-mass spectrometric (GC/NICI-MS) approach employing stable isotope dilution (6). For quantification purposes, we measure the F2-IS0P, 15-F2t-IsoP, and other F2-IsoPs that coelute with this compound. Several internal standards are available from commercial sources to quantify the IsoPs. In our... 

Mass spectrometric analysis using negative chemical ionization techniques affords greater sensitivity over electron impact methods in that a very intense ion expressing the molecular weight of the compound of interest is produced. Quantification using the stable isotope dilution method is exact and reproducible. The method does not rely on the efficiency of extraction, but rather on the ratio between the compound of interest and the internal standard, which remains constant in an individual sample. 

We have developed a very sensitive assay which can quantify both 13-cis and all trans retinoic acid in the same plasma sample. Only 1 ml of plasma is necessary for analysis, with a limit of quantification of 0.5 ng/ml. The assay is linear for both cis and trans retinoic acid, and there is virtually no interconversion of the two isomers by assay manipulations. However, the assay does slightly underestimate the amount of all trans retinoic acid present due to the differential recovery of this isomer from plasma as opposed to recovery from PBS. This will be corrected in future work by the addition of a stable isotope labelled all trans retinoic acid internal standard for quantification. 

LC-MS-MS methods for the determination of 5-HIAA in urine have been developed. one of which uses an automated solid phase extraction, isocratic LC elution, and quantification against a stable isotope-labeled internal standard.With these procedures, sample preparation is automated, chromatographic interferences are eliminated, and analytical time is reduced (2 minutes per sample). 

Two general methods utilizing the stable isotope dilution technique have been successfully used for the quantification of drugs and their metabolites by ms. The simplest, but least sensitive and specific method, is a non-chromatographic technique that utilizes chemical ionization mass spectrometry (cims) and internal standards labeled with stable isotopes. This method has been used to quantify the antiarrhythmic drugs, lidoq lne and quinidine and some of their metabolites, in human plasma samples. 

Determination of absolute quantities of peptide levels mostly employ the stable isotope dilution (SID) methodology coupled to mass spectrometric analysis. SID involves the use of stable isotope-labeled standards wherein quantification is performed by comparing the mass spectrometric profiles of analytes to their corresponding labeled standards. It is important to choose the internal standard carefully to accurately measure absolute quantities from signal intensities. 

DNA isolation from any tissue of interest after compound administration followed by (1) addition of stable isotope-labeled internal standard for quantification, (2) hydrolysis/ digestion of DNA, (3) enrichment of DNA adducts of interest (e.g., solid-phase extraction, immunoaffinity chromatography, or DNA repair enzymes),... 

Use of chemical-specific stable isotope internal standards ensures very accurate quantification, and confirmation with certainty of the chemical nature of adducts. 

The quantification of ochratoxin A, at levels within the range 0.25-10 ng/ml from wine by HPLC-fluorescence detection, was described [193]. RP-HPLC - fluorescence method for the detection of ochratoxin A in wine with a detection limit of 0.05 ng/ml was also published [194]. A stable isotope dilution assay by LC-MS/MS was developed for quantification of the ochratoxin A by using [D5]-ochratoxin A as internal standard with a low detection and quantification limits of 0.5 and 1.4 pg/kg, respectively [195]. The LC-MS/MS method (ESI and APCI) was also applied to the analysis of contaminated coffee samples by ochratoxins A and B with absolute minimum detection limit around 10-20 pg per injection. Fragment ions from the [M+H]+ and [M+Na]+ ions of... 

Since ABA is a minor component in crude extracts of plant material, extensive purification is necessary prior to identification and quantification [8,31]. The definitive method for identifying ABA as its methyl ester. Me-ABA, is combined gas chromatography-mass spectrometry (GC-MS). Negative chemical ionization causes very little fragmentation, so that the molecular ion is the base peak. This technique has been used extensively in studies with 0-labeled ABA to determine the location of the atoms within the ABA molecule [32]. GC-selected ion monitoring (SIM) is a very sensitive method for quantifying ABA, but requires ABA labeled with a stable isotope, usually H, as internal standard to compensate for losses. 

The above facts do not favour protein or peptide quantitation using MALDI-MS. Some problems are associated with MALDI-MS quantification i) low shot-to-shot reproducibility, ii) various signal suppression effects, and iii) strong influence of sample preparation and matrix crystallisation. Nevertheless, it is possible to use MALDI-MS to obtain absolute or relative quantitation. In most cases, the idea is to use an internal standard for an absolute quantitation, but this standard must have the same physico-chemical characteristics as the quantified peptide. The use of a different peptide in terms of sequence may result in different desorption and ionisation properties. Usually, the internal standard is the same peptide labelled with a stable isotope to modify slightly the mass. 

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