Species-unspecific Method

Busto et al. used synthesized Fe-labeled transferrin to determine individual transferrin isoforms in human serum samples for the species-specific mode. The stability of the prepared proteins was tested for 1 week and no iron exchange had occurred. They concluded that the results are in good agreement with other calibration methods, e.g. the species-unspecific method however, the species-spedfic mode can offer better precision. Hoppler et al. also synthesized and characterized Fe-labeled Phaseolus vulgaris ferritin for isotope dilution analysis.  

More recently, Deitrich et al. described the chemical preparation and characterization of an isotopically enriched metalloenzymes (superoxide dismutase, SOD) containing two different metal isotopes. The metal co-factors in enzyme were firstly removed and then replaced with isotopically enriched Cu and Zn. This application shows the potential of using isotopically enriched SOD for the accurate quantification of that enzyme in real samples.  

The use of species-specific spiking greatly enhances the traceability of the analytical procedure and improves the reliability of the results. On the other hand, the use of isotopically enriched metalloproteins provides a means to evaluate the analytical conditions, under which the metal center is most stable. Despite all the advantages of species-specific spiking for ICP-MS, it has to be mentioned that huge effort is necessary to produce and characterize an isotopically enriched protein spike. Therefore, this approach might be sensible in practice for the assay of only a few proteins or biomarkers.  

The method of the species-unspecific modes is well described in literature. In brief, isotope ratios are monitored during the whole chromatographic procedure and corrected by the isobaric interference, dead time, and mass bias. Then the chromatogram of isotope ratios is transformed into the chromatogram of mass flow (mass versus time) using the following isotope dilution equation  

MFs is the mass flow of the sample eluting from the column Csp is the concentration of the element in the spike (ngg ) is the density of the spike (gmL ) 

---

This technique has been used for a variety of element species, such as trimethyl- and triethyl-lead, organotins, iodine, methylmercury, and chromium. With HPLC, isotope dilution has been performed with species-specific as well as species-unspecific methods. 

Dimethyl-Tl (Me2Tl ) was determined in 500 ml sea water samples by combining Isotope Dilution Mass Spectrometry (IDMS) with a species-specific extraction method (18). A ° Tl-enriched spike solution was applied for the isotope dilution step and the production of positive thermal ions was used for mass spectrometric measurements. After species-unspecific T1 enrichment at an anion exchanger, inorganic T1 was oxidized to Tl(III) by a Br2 solution and then separated by extraction with methyl xo-butyl ketone, whereas Me2Tl remained in the aqueous phase. The detection limit for Mc2Tl is 440 pg 1 (as Tl). 

Chapters on sample introduction and hyphenated sample treatment and ICP systems have also been further updated since the last edition. No doubt that chromatographic, electrophoresis, flow injection and field flow fraction separations have extended ICP-MS (and AES) measurements as the mainstay of elanental specia-tion measurements in biological and environmental fields. Without the combination of these separation techniques and ICP measurements, elemental speciation applications would be severely hampered... if not impossible (Chapter 18). The ability to measure P and S with high sensitivity has opened up new opportunities in proteomics, for example. Species-specific and unspecific isotopic dilution (ID-MS) has been critical in quantifying speciation analysis and revealing recovery errors (Chapter 13). Species-specific techniques have been applied to identify species transformations, resulting in the development of multi-species methods whereas, hyphenated species-unspecific ICP-ID-MS determinations of heteroatoms such as sulfur have become a common quantification technique in proteomics. 

Since disturbed acid phosphatase activity has been associated with pathological conditions, the research has focused on the development of diagnostic methods for detection of activity as a marker for the onset of the disease, and in some extent to the development of inhibitors rather than activators to treat those conditions in which the increase in enzyme activity has a direct effect on the evolution of the disease. In particular, only the development of bisphospho-nate derivatives as inhibitors for tartrate-resistant acid phosphatase found their way to the market for treatment of osteoporosis [41], Typical inhibition of phosphatase activity includes anionic species such as L-(+)-tartrate, phosphate, vanadate, molybdate, and fluoride and neutral molecules such as formaldehyde. Vanadate ion,, is a competitive unspecific inhibitor for acid phosphatases by forming transition state analogs. Other oxoanions such as molybdate and tungstate also show inhibitory effects on... 

The physicochemical change of the biologically active material resulting from the interaction with the analyte must be converted into an electrical output signal by an appropriate transducer. On the one hand, unspecific, but broadly applicable transducers may be used, which indicate general parameters such as reaction enthalpy (thermistor), mass change (piezoelectric crystal), or layer thickness (reflectometry). On the other hand, a specific indication may be achieved with potentio-metric or amperometric electrodes for species such as H+, OH-, CO2, NH3, or H2O2, or with optical methods such as photometry or fluori-metry. 

Chemiluminescence in the liquid phase Chemiluminescent markers may be used to detect radical molecules like superoxide or NO. Luminol in particular reacts with NO to give a metastable intermediate, which relaxes by light emission at 427 nm. The method is sensitive, but unspecific, as the same emission is also observed with a range of other reactive oxygen and nitrogen species. In addition, the method cannot be used with optically dense samples like tissue. 

The detection method should be as species specific as possible, and ideally one would like to measure both reactant disappearance and product formation. The method must not be subject to interference from other reactants and should be applicable under a wide range of concentration conditions so that the rate law can be fiilly explored. Often there is a practical trade-off between specificity, sensitivity and reaction time. For example, NMR is quite specific but rather slow and has relatively low sensitivity, unless the system allows time for signal accumulation. Spectrophotometry in the UV and visible range often has good sensitivity and speed, but the specificity may be poor because absorbance bands are broad and intermediates may have chromophoric properties similar to those of the reactant and/or product. Vibrational spectrophotometry can be better if the IR bands are sharp, as in the case of metal carbonyls, but the solvent must be chosen to provide an appropriate spectral window. Conductivity change can be very fast but is r er unspecific, except for reactions that involve the production or consumptirm of the or OH ions, because of their unusually large specific conductivities. 

---