Isotope labeling/spiking

Stable isotope dilution mass spectrometry (IDMS) can be a definitive analytical method for very accurate concentration determination of pantothenic acid (Cornelis 2003). However, definitive accuracy of the IDMS technique requires full equilibration between endogenous natural amounts of the compound and the isotopically labelled spike, which is usually accomplished by bringing both in the same chemical form in solution. 

Stable labelled isotopes are spiked into samples before extraction and the ratio of unlabelled compound and stable labelled isotope was used to quantitate the unlabelled compound. Analysis is by high-resolution gas chromatography-mass spectrometry. Fifteen standard water samples and ten standard soil samples containing 2,4-D at known concentrations were analysed. Compound concentrations ranged from 100 to lOOOOug per kg for soil samples. Average recoveries were over 84% and method precision, given as relative standard deviation, was better than 19%. 

The Re- Os method was first applied to extraterrestrial samples in the early 1960s when Hirt et al. (1963) reported a whole-rock isochron for 14 iron meteorites that gave an age of 4 Ga. Further development of this system was hindered by several technical difficulties. Rhenium and osmium each exist in multiple oxidation states and can form a variety of chemical species, so complete digestion of the samples, which is required to chemically separate rhenium and osmium for mass spectrometry, is difficult. In addition, accurate determination of rhenium abundance and osmium isotopic composition requires spiking the samples with isotopically labeled rhenium and osmium, and equilibration of spikes and samples is challenging. A third problem is that osmium and, particularly, rhenium are very difficult to ionize as positive ions for mass spectrometry. These problems were only gradually overcome. 

The remark has been made that compounds of tin can be studied by more techniques than those of any other element. The fact that it has more stable isotopes that any other element gives it very characteristic mass spectra, and isotopic labelling can be used to interpret vibrational spectra, and for spiking samples in trace analysis two of the isotopes have spin 1/2 and are suitable for NMR spectroscopy, and their presence adds information to the ESR spectra of radical species. Further, the radioactive isotope 119mSn is appropriate for Mossbauer spectroscopy. The structural complications that are referred to in the previous chapter have therefore been investigated very thoroughly by spectroscopic and diffraction methods, and structural studies have always been prominent in organotin chemistry. 

Isocratic or gradient HPLC with MS/MS detection SPE, protein precipitation, liquid-liquid extraction, or online clean up typically using the 96-well microplate format Internal standardization based on a stable isotope-labeled analog of the analyte is prefered Accuracy three levels, five determinations each <15-20% of the spiked values... 

Soil Extraction, Soil samples were thawed at room temperature for 2 hours and ware extracted as follows 50-g aliquots of the fresh sandy loam soil ware slurried with 10 mL of organics-free water and spiked with known amounts of stable-labeled isotopes. The spike was added in 100 to 400-yL methanol aliquots to the wat soil and was allowed to equilibrate with the soil for 1 hour. Two separate aliquots were extracted for each sample. One aliquot was extracted at neutral pH to separate atrazine, lindane, and diazinon. The other aliquot was extracted at acidic pH to separate dicamba, 2,4-D, and pentachlorophenol. 

Incorporation of an isotopically labelled analogue (the spike) into the sample matrix, ideally in an identical manner to the natural compound (analyte), where further changes with time do not occur. 

Wang W, et al. Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal Chem 2003 75 4818-4826. 

It should be noted here that there are some fundamental differences between organic and inorganic ID-MS. In organic ID-MS, the isotopically enriched analog is an organic molecule that has been labeled with (usually) either H, or on a nonlabile site. In order to prevent interference from low abundance isotopomers multiple labeling is usually performed so that the mass of the labeled spike differs from the analyte by at least 3 mass units. The spike isotopes have extremely low abundances in nature (Table 1), so the labeled spike isotopomer is of extremely low abundance in the sample. Conversely,... 

Likewise, in organic ID-MS, multiple labeling of the isotopically enriched spike is usually performed so that its mass differs from the analyte by at least 3 mass units. This ensures that interference from the sample isotopomer at M +1 does not occur. 

Similarly, stock and spiking solutions of an SIS should be made entirely independently of those for the analytical standard. Note that the SIS is usuaUy available in much lower quantities than the standard analyte (particularly if it is a stable isotope labeled SIS, see Section 9.4.5), so the strategy used to prepare spiking solutions from stocks can be quite different. 

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