Consequently, few approaches describe the successful injection of crude extracts, and the majority of publications describe a sample cleanup prior to liquid chromatography with solid-phase extraction as the most efficient procedure; in particular, the use of Mycosep columns proved straightforward and efficient. 4,5,6,7,8,9
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Stable Isotope Dilution Assay
To overcome matrix effects and related quantification problems, external matrix calibration for each commodity tested has so far been recommended. This process is extremely time consuming and impractical under routine conditions, in which a variety of matrices are present every day. An alternative approach, the use of stable isotope-labeled internal standards, has recently been introduced.10
These substances are not present in real-world samples but have properties identical to the analytes. Internal standards are substances that are highly similar to the analytical target substances: Their molecular structure should be as close as possible to the target analyte, while the molecular weight is different. Within the analytical process, internal standards are added to both the calibration solutions and analytical samples. By comparing the peak area ratio of an internal standard and the analyte, the concentration of the analyte can be determined.
Ideal internal standards are isotope-marked molecules of a respective target analyte, which are usually prepared using organic synthesis by exchanging some of the hydrogen atoms with deuterium or exchanging carbon-12 with carbon-13 atoms. Physicochemical properties of such substances, especially ionization potential, are very similar to or nearly the same as their naturally occurring target analytes, but because of their higher molecular weight (due to the incorporated isotopes), distinction between the internal standard and target analyte is possible.
Considering the wide range of polarities of the analytes, the seemingly highly selective MS/MS detection could lead to the misperception that matrix interferences could be eliminated effectively and quantitative results be obtained without any cleanup and with very little chromatographic separation.
Variations during sample preparation and cleanup, as well as during ionization, are compensated for so that methods with especially high analytical accuracy and precision can be developed. Optimally, these isotope-labeled analogues must have a large enough mass difference to nullify the effect of naturally abundant heavy isotopes in the analyte. This mass difference will generally depend on the molecular weight of the analyte itself; in the case of molecules with a molecular weight range of 200 to 500, a minimum of three extra mass units might be required.
Isotope-labeled standards supplied by Biopure are fully labeled, providing an optimum mass unit difference between the labeled standard and target analyte. For example, the [13C15]-DON standard, which is available as a liquid calibrant (25mgl-1), was thoroughly characterized by Häubl and colleagues with regard to purity and isotope distribution and substitution, the latter being close to 99%.9 Fortification experiments with maize confirmed the excellent suitability of [13C15]-DON as an internal standard, indicating a correlation coefficient of 0.9977 and a recovery rate of 101% +/- 2.4%. When the same analyses were run without considering the internal standard, the correlation coefficient was 0.9974 and the recovery rate was 76% +/- 1.9%, underlining the successful compensation for losses due to sample preparation and ion suppression effects by the isotope-labeled internal standards.10,11
Direct coupling of a liquid phase separation technique such as liquid chromatography and mass spectrometry has been recognized as a powerful tool for analysis of highly complex mixtures. The main advantages include low detection limits, the ability to generate structural information, the requirement of minimal sample treatment, and the possibility to cover a wide range of analytes with different polarities.
Depending on the applied interface technique, a wide range of organic compounds can be detected and flows up to 1.5 ml/min can be handled.12 Despite their high sensitivity and selectivity, LC/MS/MS instruments are limited to some extent due to matrix-induced differences in ionization efficiencies and signal intensities between calibrants and analytes; ion suppression/enhancement due to matrix compounds entering the mass spectrometer together with the analytes also limit ruggedness and accuracy and pose a potential source of systematic errors.