Internal standard isotopes

Tebuconazole (provided by Bayer), Q -[2-(4-chlorophenyl)ethyl]-o -(l,l-dimethyl-ethyl)-li/-l,2,4-triazole-l-ethanol. Molar mass 307.8, (M- -H)+ ion observed at approximately m/z 308.1 [liquid chromatography/mass spectrometry (LC/MS)] Tebuconazole-fnflzoZe-i,2,4-- fV3 (provided in acetonitrile solution by Bayer), [ NsJtebuconazole stable-isotope internal standard, o -[2-(4-chlorophenyl)ethyl]-Q -(l,l-dimethylethyl)-li/- A3-l,2,4-triazole-l-ethanol. Molar mass 310.8, (M -I- H)+ ion observed at approximately m/z 311.1 (LC/MS)... 

Cyfluthrin-met/ty/-fi(6 (provided in acetonitrile solution by Bayer Corp.) Cyfluthrin stable-isotope internal standard (IS), Cyclopropanecarboxylic acid, 3-(2,2-dichloroethenyl)-2,2-dimethyl-, cyano(4-fluoro-3-Phenoxyphenyl)methyl ester-met/tyWe, molecular formula C22H12D6CI2FNO3, molar mass 440.3, ion observed at 213.0 (GC/MS)... 

H6]-Leukotriene A4 methyl ester, 17, has been synthesized14 by Wittig olefination of epoxy dienal 18 with the key reagent 3,4,6,6,7,7-[2H6]-(Z)-(3-nonen-1-yl)triphenylphosphonium iodide, 19 (equation 9). 17 is employed as stable isotope internal standard for the MS trace analysis of eicosanoids15-17. 

Homogenization with HjO Addition of HAc centrifugation filtration Homogenization with CHES-HEPES buffer addition of isotope internal standard filtration... 

The right internal standard should have similar chemical and physical properties to match those of the analyte of interest. In LC-MS and LC-MS/MS assays, the coelution of the internal standard represent the ideal situation. Therefore, both structural analogs and stable labeled isotope forms of the analyte can be used. Stable labeled isotope internal standards are an ideal choice of every mass spectrometric-based assay because they offer the highest precision. However, not all the stable labeled isotope forms of the analyte behave in the same way when used as an internal standard. Actually, the analog compounds may behave better than the wrongly labeled isotope internal standards. 

The disadvantages of stable labeled isotope internal standard are its cost and availability. They are very expensive because they often require custom synthesis. 

Minimum sample degradation can be controlled by the addition of stable labeled isotope internal standard at time of collection [3,5,19]. 

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. 

Stable isotope internal standard stock solutions ... 

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

It is important to first note that many classical clinical assays have traditionally measured one metabolite to detect one disease. Consequently, many of the rules of method validation were designed around this premise. MS/MS, as originally designed, detected two classes of compounds, amino acids and acylcamitines, in four to five different MS methods (known as scan modes such as neutral loss, precursor ion, or selected reaction monitoring), for approximately 500 distinct masses, more than 70 known compounds, and 20-30 stable isotope internal standards. How then did one approach such a complicated validation to gain acceptance as a reliable, useful method The answer is quite simple - start simply and compare to what was already established. 

Estrogens in clinical chemistry RIA, GC-MS/MS, and LC-MS/MS analyses of estrogens in serum and plasma isotope internal standard sample derivatization ionization modes and sensitivities of GC-MS/MS and LC-MS/MS. [4]... 

Targeted analysis refers to metabolome analysis that targets one, or a few metabolites, and typically uses an internal standard for quantitation. The most common method is isotope dilution mass spectrometry (IDMS) [34], which relies on the use of stable isotope internal standards to enable the absolute quantitation of metabolites. This method has proven highly effective and has been successfully used in numerous studies. 

For HPLC MS/MS assays the use of stable isotope labeled internal standards is by far the best method to overcome any potential matrix effects and random variation in the MS/MS detector. If for any reason this stable isotope internal standard is not available, an analog compound with a mass different from the analyte can also be used. The chromatographic retention time of the internal standard, however, should be as close as possible to the retention time of the analyte. This ensures, that time dependent random variation in the ionization chamber, or whereever else in the MS/MS detector, are compensated by the internal standard. In a toxicokinetic assay described by Chi et al. (2003), for example, an internal standard was used which showed the same retention time as the analyte. 

Bravo R, DriskeU WJ, Whitehead, Jr RD, Needham LL, Barr DB. Quantitation of diaUcyl phosphate metabolites of organophosphate pesticides in human urine using GC-MS-MS with isotopic internal standards. J Anal Toxicol 2002 26 245-52. 

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

MS or MS-MS coupled with GC or HPLC has been u.sed for the determination of DAPs in urine (Bravo et al., 2002 Hardi and Angercr, 2000 Hernandez et al., 2004 Oglobine etal.. 2001). The use of GC/MS or GC/MS-MS requires alkylation of DAPs in order to render them volatile for GC separation. HPLC coupled with MS or MS-MS has the advantage that DAPs need not be alkylated. Fimhermore. the use of labeled (isotopic) internal standards compensates for the losses in recovery and improves precision. However, the use of labeled internal standards is very expensive since these compounds have to be custom synthesized. 

Selection of the most appropriate isotopic internal standard (often termed the spike and in this guide genetically referred to as the isotopic analogue)... 

Other problems, not necessarily affecting different procedures to the same extent, include lengthy cleanups and gas chromatographic separations, unavailability of isomeric TCDD standards, and impurities in isotopic internal standards. The choice of different signal-to-noise ratios by different laboratories affects the detection and measurement limits. 

Derivatization and nonstoichiometric reactions Ideally, only isotopic internal standards should be used, as it is naive to expect two chemically distinct entities to react identically. The causes of the imprecision should be investigated and the reaction conditions modified to make them suitably robust rather than rely on the inclusion of a second compound. However, potential problems with isotope internal standards need to be considered, particularly if the bond(s) linking the isotope to the rest of the molecule are involved in the reaction. It may be necessary use alternative sites of labeling. 

The opinions written in the workshop conference report (Viswanathan 2007) represent a practical approach to assessing matrix effects with certain assumptions, i.e. using multiple individual lots of matrix spiked with known amounts of analyte will provide a good indication for matrix effects for the method in general, and stable isotope internal standards will provided significant compensation for matrix effects that will result in lower variability of response and better method reproducibility. A further discussion of how to assess and minimize matrix effects as part of the development process can be found in Chapter 9, and the guidelines discussed above can be incorporated into... 

Pu5/92138 issued by AERE, Harwell (Hamilton et al. 1989). The isotope fractionation at each step is corrected by reference to the certified isotope ratios. The idea was further developed by Dubois et al. into a dynamic multidetection measurement mode (Dubois et al. 1989), which practically eliminates mass fractionation effects and possible mismatches of cup efficiencies with a 2-isotope internal standard and MIC/TIMS. According to the results presented, precisions and accuracies of 0.01 % are achievable with this procedure. An ultimate refinement has been introduced by performing total evaporation measurements with peak tailing correction in dynamic multicollection mode, using a MIC/TIMS with magnetic sector equipped with a dispersion quadrupole (Goldberg et al. 2002 Richter and Goldberg 2003). 

A rigorous approach to evaluate the memory effect is the sequential analyses of natural sample, enriched isotope (internal standard or tracer), and again the natural sample. As shown in Fig. 1, no measurable memory effect was observed for Hg [50], a small memory effect was seen for Cd... 

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