Off-line sampling

In order to reduce or eliminate off-line sample preparation, multidimensional chromatographic techniques have been employed in these difficult analyses. LC-GC has been employed in numerous applications that involve the analysis of poisonous compounds or metabolites from biological matrices such as fats and tissues, while GC-GC has been employed for complex samples, such as arson propellants and for samples in which special selectivity, such as chiral recognition, is required. Other techniques include on-line sample preparation methods, such as supercritical fluid extraction (SFE)-GC and LC-GC-GC. In many of these applications, the chromatographic method is coupled to mass spectrometry or another spectrometiic detector for final confirmation of the analyte identity, as required by many courts of law. 

Direct injections using RAM or TFC have simplified sample preparation and increased throughput. Matrix ion suppression was greatly reduced or eliminated in several cases compared with traditional off-line sample cleanup procedures such as PPT, SPE, and LLE. Method development time was minimized with generic methods15 that suit most applications. Detailed applications can be found in a recent review.8... 

The most common (off-line) sample preparation procedures after protein precipitation are solid phase extraction and liquid-liquid extraction. Multiple vendors and available chemistries utilize 96-well plates for solid phase extraction systems and liquid-liquid extraction procedures. Both extraction process can prepare samples for HPLC/MS/MS assay. Jemal et al.110 compared liquid-liquid extraction in a 96-well plate to semi-automated solid phase extraction in a 96-well plate for a carboxylic acid containing analyte in a human plasma matrix and reported that both clean-up procedures worked well. Yang et al.111 112 described two validated methods for compounds in plasma using semi-automated 96-well plate solid phase extraction procedures. Zimmer et al.113 compared solid phase extraction and liquid-liquid extraction to a turbulent flow chromatography clean-up for two test compounds in plasma all three clean-up approaches led to HPLC/MS/MS assays that met GLP requirements. 

Gas chromatography (GC) Hewtili-Pmrfwttf 8890 Online reaction study, off-line sample analysis Trained Free... 

Off-line sample pretreatment in ion chromatography most often involves matrix elimination using... 

Sample pretreatment is useful when a mass spectrometer with atmospheric pressure electrospray ionization (ESI/MS) is used as the detector for ion chromatography. High ionic strength matrices are known to suppress analyte ionization and cause poor reproducibility in ESI/MS [40]. We investigated the use of off-line sample pretreatment to remove chloride in order to improve sensitivity in... 

In the case of off-line sample preparation methods, clean up is required in samples having colors and other interfering impurities. Biological and environmental samples contain thousands of other substances as impurities and these were extracted along with the analytes of interest. Due to the similar properties of the co-extractives, they usually interfere in the analyses of the... 

Hg(II). This conversion is usually on-line, but in an effort to analyze low levels of mercury species, some workers have developed on- and off-line sample preconcentration methods.108110... 

The major disadvantage of column switching is that some excipients may become irreversibly bound to the sorbents, consequently requiring relatively frequent changes in the sorbents. Under these circumstances, the off-line sample pretreatment cleanup procedures described in Chapter 2 should be used. 

Jansen and co-workers [86] have evaluated temperature-controlled outgassing processes of plastics and rubbers using both off-line and on-line TD-GC-FTIR-MS. Decomposition of polyesterurethanes by means of TG-Tenax off-line sampling followed by TD-GC-FTIR-MS revealed C02, H20, tetrahydrofurane, cyclopentanone, dicarbonic acid, aliphatic diols and esters [86]. The same authors have also described the detection of polychlorinated biphenyls (PCB) in 2,4-dichlorobenzoylperoxide cured silicone rubbers after outgassing products of a rubber silicone part obtained after desorption for 10 minutes at 200 °C in the thermal desorption cold-trap and subsequent analysis by means of TD-GC-MS. Using a mass range of 290-294 Da the MS can be used as a selective detector for these substances. 

Fig. 10.6. Relalion.ship belwecn the theoretical plate height H) and eluent front velocity (u) asing OPLC with forced flow of the mobile phase / = fully on-line OPLC 2 = on-line sample application, off-line detection J = off-line sample application, on line detection 4 = fully off-line OPLC. Optimum of flow velocity can be reached by using fully off-line OPLC. with a set-up for forced flow of the mobile phase. Reproduced by permission from Ref. [80].

Summary The use of the on-line FT-Raman spectroscopy for monitoring a multi-step hydrosilylation reaction combines all the advantages of an on-line analytical tool (like real time measuring results, a direct view into the reaction, and no off-line sample collection) with the requirements for the application of technology in production plants, e. g., low calibration effort within a wide temperature range, stable calibration, simple system handling for the operator, small sized equipment at the reaction vessel, and no contact with the reaction media. 

Proper sample pretreatment methods are important to reduce or eliminate matrix effects. In this section, advances in sample pretreatment methods in relation to LC-MS quantitative bioanalysis are briefly reviewed (see also Ch. 1.5). Contrary to environmental analysis, where on-line sample pretreatment is often used (Ch. 7.3.2), off-line sample pretreatment appears to be preferred in quantitative bioanalysis. This is partly due to the composition of biological samples, where the presence of especially proteins may cause clogging of the SPE columns, cartridges or disks used, and partly due to the fact that decoupling sample pretreatment and LC-MS analysis generally allows for a higher sample throughput. 

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

Many different sample preparation techniques are available to the drug discovery scientist. Off-line sample preparation procedures include protein precipitation, filtration, dilution followed by injection, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Typically, these procedures are performed in an automated, high-throughput mode that features a 96-well plate format. Online sample preparation procedures include SPE and turbulent flow chromatography (TFC) with conventional chromatographic media or restricted access media (RAM). These online approaches are often simple and easy to automate. 

With the on-line approach, the sample is continuously delivered to the vacuum in real time. Off-line sample introduction entails the running of microfluidic processes on the chip with later MS analysis. Therefore, the introduction of samples into the ion source generally requires breaking the ion source vacuum. 

---