Library sample

Figure 4.93. PCA of TEA SI MCA library samples (O) with unknowns (labeled with numbers). The first two principal components are used to make the TEA SIMCA model.

For example, it makes sense that the library has a reference to the compound samples it contains. Also, each compound sample should have a reference to the library to which it belongs. Multiplicity specifies the number of objects of one class that can be associated to an object of another class. For example, a compound sample can have only one notebook, and so the multiplicity is 1 at the Notebook end of the Sample-Notebook association. A library can have 1 to many compound samples, and so the multiplicity is l..n at the Sample end of the Library-Sample association, and so on. If multiplicity is obvious during the analysis phase, then specify it. Otherwise, defer this decision to the design phase. 

A discrete library is a set of compounds that are obtained as individuals at the end of the library synthesis. Parallel synthesis leads to a discrete library by simultaneous addition of reactants in different reaction vessels and parallel handling of each library sample (see the example in Fig. 4.2, where 15 discretes are prepared from the common intermediate A with two parallel reaction steps). Conversely, a pool library is a set of compounds that are obtained as mixtures, or library pools, at the end of the synthesis. Mix and split (or divide and recombine) is the process leading to an SP pool library... 

The structural database must be linked to a relational database (42), which contains all the generated information for a discrete or a pool library sample. The relational database includes structural information (MW, molecular formula), physicochemical properties (log P, solubility, p/fa), analytical results (peak values or spectra IDs from which the spectra can be found), and activity data from primary and secondary... 

Figure 8 (a) Negative ion ESI spectrum of the 36-component library sample showing the ppm differences between theoretical and found m/z values for the assigned peaks, (b) Negative ion ESI spectrum of the 120-component library sample resolving nominally isobaric peaks. (Reprinted from Ref. 91.)... 

There is another reason to favor smaller molecules. Screeiung a library is an experiment in which chemical space is sampled with regard to the ability to bind to the target of interest. The better a given library samples chemical space, the higher the probability of finding all possible... 

FIA is the simplest form of sample introduction into the mass spectrometer, and this injection format has been widely used in the analysis of combinatorial library samples. This technique offers the highest throughput combined with ease of use and facile automation. Richmond et al. [67-69] reported methods to minimize sample carryover for the FIA-MS analysis of combinatorial libraries. Samples were sorted before the analysis to maximize the molecular-weight difference between samples in the analysis queue and to minimize the conditions where consecutively measured wells contain samples similar to building blocks. Cycle times of less than a minute were reported with a carryover of 0.01%. A software appUcation was developed to automatically report the sample purity and calculate sample carryover by an automatic spectrum comparison method [70,71]. A quasi-molecular ion discovery feature was also implemented [72] in the automated data-processing program. Automated FIA-MS analysis and reporting were also used in the analysis of fractions from the purification of combinatorial libraries [73]. Whalen et al. developed software to allow automated FIA-MS analysis from 96-well plates [74]. The system optimizes the interface for mass spectrometry and MS/MS conditions, and reports the results in an unattended fashion. 

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

Reported bioanalytical applications that use SS-LLE products include both tlie cartridge (tube) format and the microplate format. Some of these applications include the determination of mexiletine [44], amiodarone [45] and other antiarrhythmic drugs [46], proxyphylline [47], 16(3-hydroxystanozolol [48], dextromethorphan [49], and simvastatin [50] from biological fluids. Microplate applications for SS-LLE include a crude purification of crude combinatorial library samples [51], carboxylic acid-based matrix metallo-protease inhibitors [52], and a p3-adrenergic receptor agonist [53]. 

Finally, the hit quality index does not provide any absolute measure of the probability that the sample actually is the same as the library sample. The arbitrary scale of the hit quality values (0-1) does not give a very good statistical measure of the similarity of the spectra. In short, using only a single training spectrum to represent all possible samples in the future does not give the analyst any statistical assurance that the spectra are truly the same or different. It provides only a relative measure for all the library samples. For anyone who has tried simple library search techniques for spectrally similar samples, this result is all too obvious. 

To understand this process, consider as an example that an unknown is a 50/50 mixture of library sample 6 and 37. Under ideal conditions the algorithm would predict that the Cl for the unknown was 0.5 for 6 and 0.5 for 37 all of the other composition indices would be close to 0.0. In real unknown mixture spectra, the prediction is not this definitive, but it works well considering its simplicity. 

At the end of each day, all the scans were subjected to qualitative analysis using the vendor s software. After evaluating various data treatments, first derivatives were used for all library entries and all predictions. The resulting printed report contained the date, time of each scan, sample name, sample identification when compared with the stored library, and Mahalanobis distance from the library sample. [Editor s note if the reader doesn t understand Mahalanobis distances, refer to Chapter 15 in Part in.]... 

Fig. 2. Schematic representation of automated 96-well ion-exchange solid-phase extraction for the removal of anionic components snch as carboxylic acids from crude library samples.

HPLC assay The purity of the final combinatorial products is determined by the aforementioned gradient HPLC method. The library samples before and after extraction are injected onto the HPLC system. The product and impurity peak areas are then compared to assess the product purity and recovery. The product purity is determined by the percent peak area of the product of interest in the post-extraction sample solution. The product recovery and the removal of the excess reagents, by-products, or impurities are evaluated by the ratio of the peak areas of the respective component before and after extraction. 

For the library samples that contain very polar by-products, excess reagents, or impurities, an ion-pair reagent may be added to the mobile phase to improve retention of the polar components on the reversed-phase HPLC column. For small polar basic components such as amines, 0.1-1% of heptafluorobutyric acid can be added into the mobile phase containing acetonitrile or methanol in water. For small polar acidic components such as carboxylic acids, 5-20 mM of tetrabutyl-ammonium dihydrogenphosphate can be added into the mobile phase containing acetonitrile or methanol in 50 mM phosphate buffer (pH 7.4). 

Most commercial FTIR software packages come equipped with a library searching capability, or it can be purchased for an additional fee. When a spectral library search is performed, the unknown spectrum is compared to each spectrum in the libraries selected. Thus, if there are 1000 spectra in a library, 1000 comparisons are performed. As a result of each comparison a number called the hit quality index (HQI) is calculated, which is a numerical measure of the similarity between two spectra. In an ideal world the library search will turn up a spectrum similar to the unknown spectrum. It is then assumed the unknown sample is chemically similar to the library sample. This way known spectra can be used to identify unknown spectra. Library searching is so useful in identifying unknowns and interpreting mixture spectra that I encourage all FTIR users to have access to library searching capabilities. 

High-performance liquid chromatography (HPLC)-grade chloroform and methanol were obtained from Fisher Scientific Co. Peptide lipid library samples were dissolved in a mixed solvent of chloroform, methanol and CF3COOH (5 1 0.01, v/v/v) to a concentration of 1.0 mM. The injected volume was 40 )u-L for all samples. After spreading the sample, the solvent was allowed to evaporate for 15 min. The water used for the monolayer study was purified by a Modulab 2020 water purification system (Continental Water Systems Corp., San Antonio, TX). The water had a resistance of 18 MH cm and a surface tension of 72.6 mN/m at 20°C. The D-maltose, d-glucose, and sucrose used for subphase preparation were pmchased from Aldrich Chemical Co. and were dissolved in deionized water to a concentration of 10 mM. All these subphases had a pH of 5.8. The compression rate was set at 4 A molecule min for the smface pressme-area isotherm measmements. 

This research is currently in a very early stage. Much deeper and further investigation is needed. Above all, the feasibility of this novel combinatorial surface chemistry technique needs further experimental evidence. The study of Langmuir monolayers made from lipid library samples is unprecedented in itself and, therefore, requires considerable work in this respect. For ex-... 

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