Unlabeled compound

An isotope that is used extensively is deuterium (heavy hydrogen), often in the form of a deuteromethyl (-CD3) group. The molecular weight of this compound is thus three Da higher than the unlabelled precursor and this is often sufficient to ensure that the ions in the molecular ion region of the unlabelled compound do not occur at the same m/z ratios as those from the labelled molecule. 

When [ H]-labeled precursors are employed the resulting compounds can be used as internal standards for analysis, especially by utilization of mass spectrometric methods. Appropriate deuterated standards are shown in Fig. 7. The introduction of deuterium into the A9-THC precursors can be done with Grignard reagents such as C[ H3]MgI or reducing substances such as LiAl[ H4]. The general procedures for the synthesis with these [ Hj-labeled precursors are the same as described above for the unlabeled compounds [76,78]. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

Plasma levels of diisopropyl methylphosphonate were measured in a single lactating Jersey cow after the sixth day of diisopropyl methylphosphonate oral administration (10 mg/kg/day) by gelatin capsule (Ivie 1980). For the first 5 days the cow was given unlabeled compound and fed hay ad libitum. The diisopropyl methylphosphonate administered on the 6th day was labeled with carbon 14. Based on measurements of label in the plasma, absorption in the cow paralleled that in rats and dogs, with the highest concentration detected in the plasma at 2 hours after administration. 

Electron ionization is a perfect method for the analysis of labeled molecules as in this case ion-molecular reactions are suppressed. It is better to use for the calculations the most intense spectral peaks with the highest m/z values. Molecular ion is the best choice. However, if notable [M + H]+ or [M — H]+ peaks are present in the spectrum of the unlabeled compound the correct calculation will be problematic. To eliminate [M + H]+ peaks it is helpful to record a spectrum with the minimum quantity of sample. To consider interference with [M — H]+ ions one should know from what position the hydrogen atom is lost and whether deuterium could be in this position. 

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%. 

Form Figure 32.2, it is quite evident that the percentage of radioactive compound bound A decreases with the continual addition of unlabelled compound A. 

Figure 32.3, depicts the plotting of the percentage inhibition of labelled compound binding A against the continual addition of unlabelled compound A thereby giving rise to a straight line. 

Isotope dilution mass spectrometry (IDMS) is another method to overcome the problem of sample recovery [370-372]. The 13C-labeled isotope of the analyte is added to the sample at the commencement of the analysis and the ratio of the labeled and unlabeled compound is measured by MS. This technique eliminates the need for recovery measurements and automatically accounts for any losses in the determination [373]. The two major limitations of this method are the cost and availability of the labeled compounds and the need to use the MS as a detector. 

Isotope fractionation between the vapor phase and the dissolved aqueous phase has been studied only for toluene and trichloroethylene (carbon only [545, 690]). Fractionation associated with adsorption has been quantified only for toluene in regard to sample extraction using a poly(dimethylsilo-xane)-coated solid-phase microextraction fiber [373] and qualified for benzene, toluene, and ethylbenzene based on high-pressure liquid chromatography analyses of isotopically labeled and unlabeled compounds (carbon and hydrogen [692]). Isotope fractionation associated with the reductive dechlorination of chlorinated ethylenes by zero-valent iron and zinc has been... 

The solution phase synthesis of peptides often results in mixtures of labelled, unlabelled compounds along with other impurities and requires extensive purification. A route using a solid phase approach [polyethylene glycol-poly-styrene PEG-PS derivatised with a xanthen-2-oxovalerate (XAL) linker] was recently developed [193] (Scheme 44). 

The biotransformation of 1,1,2,2-tetrachloroethane was studied in rats and mice using [ C]l,l,2,2-tetrachloroethane. The metabolic disposition study was conducted after oral administration of the unlabelled compound on five days per week for four weeks, followed by a single dose of the radiolabelled compound to simulate conditions of a bioassay for carcinogenicity testing. After oral administration of 0.59 mmol/kg bw (98.5 mg/kg bw) [ C]l,l,2,2-tetrachloroethane to rats and 1.19 mmol/kg bw (198.7 mg/kg bw) to mice, 7% and 9.7% of the administered radioactivity were recovered in the expired air of rats and... 

Sometimes, however, the exact arrangement of the carbon atoms is not immediately clear from the standard spectra and, of course, we cannot identify the quaternary carbon atoms from an HMQC spectrum. Tn these cases, further information is required about atom connectivities, and as C is so low in natural abundance, a correlation experiment is not realistically possible with unlabelled compounds. It is possible, however, to detect couplings over... 

After the incubation period (e.g. 30 min), the tubes are washed several times with water. Only bound molecules will remain in the tubes. The amount of enzyme-conjugated compound bound to the wall (i.e. to the antibody) will increase accordingly with decreasing amounts of free, unlabelled compound in the sample. 

Isotope dilution analysis permits one to determine the purity of a radiochemical. Compound X, molecular weight of 150 (specific activity 1.0 mCi/mmol), was checked for purity by carefully weighing 1.5 mg of the radiochemical and mixing with 1000 mg of unlabeled compound X and recrystallizing until a... 

Choose selective mass traces of labeled and unlabeled compound for subsequent quantification using single ion monitoring (SIM mode). 

To obtain the calibration curve, the amount ratio of unlabeled compound/labeled compound (x-axis) is plotted against the ratio of peak area of the mass trace of the unlabeled... 

Quantitation using unlabeled compounds as internal standards and GC-FID detection lacks the high sensitivity and the high selectivity required for aroma compounds present in the ppb level. For chemically stable compounds and those in higher concentration (i.e., >1000 ppb), however, this method gives reliable data. In all other cases, using isotope labeled compounds as internal standards is the method of choice if the they are available. 

Pulse-chase. An experiment in which a short labeling period is followed by the addition of an excess of the same, unlabeled compound to dilute out the labeled material. 

Isotope ratio is measured as the ratio of the area of the primary ion of the unlabeled compound to that of the labeled compound. When the area is zero, it is assigned a value of 1. The retention times of the analytes in most cases are the same as that of their labeled analogs. The isotope can be calculated from the extracted ion current profile (EICP) areas. An example of EICP for benzene, benzene-d6, and a mixture of benzene and benzene-d6 is presented inFigure 1.4.3. Calculation to determine the RR is given below ... 

Compounds with the same mode of action interact with the same binding site on the protein. Triazines and ureas, as well as the other compounds listed in Figure 8.1, displace plastoquinone QB. Therefore, they also displace each other from the target site in PS II, and their inhibitory potency can be evaluated by the procedure introduced by Tischer and Strotmann (1977). This is experimentally followed with a radioactive derivative in which a 14C labeled triazine is bound to the target. The radioactivity will be diluted out of this site by an unlabeled compound of similar potency and mode of action. This method does not require measuring photosynthetic activity, but does require a structurally and functionally intact PS II because binding efficiency is easily lost by improper handling of the membrane. 

Fig. 11. Effect of increasing concentrations of EM-652, E2, ICI 182,780, droloxifene, ICI 164,384, and toremifene on [3H] l7(3-estradiol (E2) binding to the rat uterine estrogen receptor. The incubation was performed with 5 nM [3H] l7(3-estradiol for 2 hours at room temperature in the presence or absence of the indicated concentrations of unlabeled compounds (Martel et al., 1998a).

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