Accuracy experiment internal normalization

McArthur et al. (Ml) and others have demonstrated that extracts prepared by the tannic acid procedure are less toxic to experimental animals than those obtained by most other techniques including the variants of the kaolin-acetone method. Herbst et al. studied the accuracy of the tannic acid method by conducting recovery experiments involving the addition of the second International Reference Preparation for human menopausal gonadotropin (second IRP-HMG) to pools of normal male urine. They found that approximately 100% of the LH activity, but only 50% of the FSH activity, was recovered (H5). 

MS, MS/MS and MS experiments were performed with a LTQ-Orbitrap (Thermo Fisher Scientific Inc., San Jose, CA, USA) mass spectrometer equipped with a Triversa Nanomate (Advion Biosciences Inc., Ithaca, NY, USA). Compound solutions were infused with a nanospray chip. The mass spectrometer was first calibrated externally with a mixture containing caffeine, L-methionyl-arginyl-phenylalanyl-alanine (MRFA) and Ultramark 1621 in ACN, MeOH, HjO, acetic acid. Sub-ppm mass accuracy was finally achieved using an internal calibration (lock mass) in both MS and MS/MS mode. The resolution of Orbitrap MS was set to 100,000 (FWHM) at m/z 400. Electrospray ionization (ESI) was used. For MS/MS experiments, an isolation width of 1.5 Da was used. The normalized collision energy was set to the value when the precursor ion was exhausted. Helium was used as the collision gas. 

The next section deals with method validation of quantitative TLC methods. Two questions should, however, be answered prior to discussing the validation experiments namely, whether the statistical evaluation of data elements, such as precision, accuracy, and reproducibility should be calculated on the basis of measured peak heights or peak areas, and whether the internal or external standard methods, or area normalization should be used to yield quantitative results for the assay. Without going into detail, the most important advantages and limitations of peak height and peak area measurements, and those of the different methods of quantification are summarized in Table 4. 

BSE methods enjoy a considerable advantage in generality over current numerical methods, with respect both to the molecules which can be treated and the complexity of the HF approximation which can be employed. There is much recent progress toward developing methods which scale well with problem size (see linear Scaling Methods for Electronic Structure Calculations). For an insensitive molecular property in a normal (usually ground) electronic state where the accuracy can be estimated from experience, BSE methods are more efficient for moderate accuracy, perhaps even high accuracy in some cases. However, the lack of an internal error estimate is a serious handicap, especially in abnormal and/or unfamiliar cases. 

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