Offline analysis techniques

At last, the double focusing sector field mass spectrometer (EBQjQ2) should briefly be addressed. This mass analyser represents a highly sophisticated early design that has rarely been used for routine analysis especially for quantification. For detailed information on the functional principle the reader is referred to respective textbooks on mass spectrometry. We mention this technique as Kajbaf et al. used EBQjQ2 for detection and identification of the QTA cimetropium and its biotransformation products from liver microsomal mixtures in offline analysis of HPLC fractions after FAB ionization [23, 62] (Table 7). 

More success was obtained using on-line solid phase extraction before offline analysis by capillary electrophoresis with matrix-assisted laser-desorption ionization (MALDI) MS [18]. Because microdialysis is a method used for analysis of small molecules and peptides, MALDI is not used frequently with microdia lysis sampling. Another disadvantage is that MALDI cannot be used on-line with the separation because it is a vacuum ionization technique. However, if the pep-tide is large enough (0 1000 Da) MALDI can be useful. For the analysis of peptides in dialysate an appropriate separation is important before mass spectrometric detection. In a comparison with direct sampling of dialysate in MALDI, capillary electrophoresis provides the high efficiency separations necessary to resolve all... 

The MALDI technique is considered an offline ionization technique because the sample is purified, deposited, and dried on the sample plate before the analysis. 

In most cases, the investigator should compare the results of the microchip analysis with those an offline standard technique (CE high-performance liquid chromatography, HPLC) for method validation. Control microdialysate samples must be collected, particularly for in vivo experiments that involve injection of a compound or drug. Such control samples can help identify or eliminate peaks from the microchip analysis. In many cases it is useful to incorporate an internal standard into the sampling process to correct for any changes in the recovery, reaction chemistry or with the on-chip injection process. 

The offline analysis of selected metabolites of the cells during a bioprocess can be performed by MS, NMR spectroscopy, or chromatographic separation procedures. The NMR technique is based on the specific response of atomic nuclei that are exposed to a magnetic field. This technique is able to resolve the structures of small biomolecules and thus to determine selected metabolites during a bioprocess or for metabolic flux analyses. Advanced MS techniques allow a high throughput analysis of complex mixtures of metabolites by the determination of the specific masses of the metabolites. 

The expression analysis by means of optical DNA chips is only useful for an offline analysis of selected critical bioprocess-relevant genes. The analysis time for the mRNA by these approaches from early cell disruption and RNA isolation to final data analysis lasts several hours. Alternative shorter and less labor-intensive techniques have been suggested and are... 

TLC plates are of particular interest as substrates for spectroscopy (i) as a storage device for offline spectroscopic analysis (ii) for efficient in situ detection and identification and (iii) for exploitation of spectroscopic techniques that cannot be used in HPLC. Thin-layer chromatography combined with HR MAS (NMR) can be used for compound identification without the need for elution from the stationary phase [413]. Recently also TLC-XRF was found suitable for in situ TLC imaging of elements [414]. The combination... 

A variety of online solid extraction devices and applications have been developed for bioanalysis. Many are easy to build in laboratories or commercially available. Unlike offline methods, minimal operator intervention is needed for daily sample analysis after online applications are set up, so the approach is both labor- and cost-effective. The technique can also minimize errors arising from manual operations, eliminate potential inconsistencies caused by different operators, and provide accessibility of LC/MS/MS applications to laboratories that have minimal analytical expertise. 

One important area of stable isotope ratio measurements (SIRMS) is the analysis of gases using offline or online techniques (see also the discussion in Section 8.3.). 

Long-lived radionuclides occur at extremely low concentrations, especially in environmental samples, therefore several authors have proposed matrix separation and enrichment of the analytes before analysis.21,24,26,3 39 Radiochemical methods often require very careful and time consuming separation and enrichment processes and measurement procedures of a-, (3- and -emitting radioactive species at the trace and ultratrace level using conventional radioanalytical techniques 40-43 Trace/matrix separation, which is performed offline or online in order to avoid possible isobaric interferences, matrix effects and to reduce the detection limits for the determination of long-lived radionuclides, is also advantageous before ICP-MS measurements as the most widely applied mass spectrometric technique. 

Of more immediate interest are approaches that permit offline TLC-MS in which the spots are scraped out from the layer and the analytes are either extracted from the sorbent to be transferred to the mass spectrometer as discrete samples or are introduced without sorbent removal into the spectrometer on a direct insertion probe (51). TLC-MS quantification and confirmation efficiency can be further enhanced by submitting the TLC extract to an additional chromatographic separation using a different technique prior to the final MS analysis. Advantages of this approach over direct TLC-MS include extra cleanup through the additional chromatographic separation (52). This has been realized in the TLC-GC-MS analysis of eggs and meat for chloramphenicol residues (49). 

The number of detectors that are sensitive and selective enough to be applied online with LC is limited because the solvents used are not compatible, as in the case of immunochemical detection after reversed- or normal-phase LC. The technology of coupling is still under development and not yet available in a large number of laboratories not specialized in techniques such as LC-MS. Therefore, LC separations are frequently followed by offline detection. Confirmatory analysis of suspected liquid chromatographic peaks can be made possible by coupling liquid chromatography with mass spectrometry. Atmospheric-pressure chemical ionization LC-MS has been employed for the identification of six steroid hormones in bovine tissues (448). 

There are cases where HPLC separation is performed not in order to quantify the alcohols but as a technique for the purification of the analytes to be subjected to further instrumental analysis. This is the case, for example, with the identification and determination of the structure of an abscisic acid in starfruit extract (Averrhoa carambola L.). The separation and purification of the analytes was carried out also with HPLC using a mobile phase of diethyl ether, whereas the structure was elucidated by H and UC-NMR (6). In a similar way, to separate the sterols and alkanols from the unsaponifiable matter from olive oils on a silica column, a gradient composed of hexane/diethyl ether was chosen in an offline system (7), whereas an online HPLC-HRGC system uses as its mobile phase hexane/isopropanol (8). 

Semipreparative HPLC has been employed to obtain a vitamin D-rich fraction of the unsaponifiable matter for subsequent quantitative HPLC. Combinations of chromatographic modes used for offline semipreparative and quantitative analysis have included polar bonded-phase/adsorption (211,212), reversed-phase/adsorption (194,213), and adsorption/reversed-phase (70,125). An online two-dimensional HPLC technique using two polar bonded-phase columns has also been described (214). 

LC-MS/MS techniques moved the bottleneck for analyzing NCEs from sample analysis to sample preparation. Several methodologies have been developed to reduce sample preparation time. Online extraction methods are becoming more popular and have many advantages over offline techniques. For example, online sample preparation is easier to automate and minimizes recovery issues. 

Samples rarely come in a form that can be injected directly into the instrument some form of sample preparation usually is required. Sample preparation includes any manipulation of the sample prior to analysis, including techniques such as weighing, dilution, concentration, filtration, centrifugation, and liquid- or solid-phase extraction. Sample preparation can be performed either on-line or off-line, but it is usually performed offline. Off-line preparation can be time-consuming and tedious, and the more steps that are required, the more susceptible the analytical method is to operator error and irreproducibility. 

Another advanced and novel sample preparation technique is online solid phase extraction (online SPE). Although the extraction mechanism is as the same as traditional offline SPE, online SPE offers several advantages. Because sample preparation is carried out during analysis, it eliminates the time needed for sample preparation thus increases throughput significantly. Additionally, because the entire sample is eluted to the LC-MS/MS system, it may increase assay sensitivity. Finally, because there is no manual extraction involved, it may reduce human error, potential contamination, and inconsistent recovery. 

Analytical methods for the determination of one antidepressant and/or its metabolite(s) were usually performed in isocratic mode, with total run times from seconds to a few minutes. However, as previously mentioned, multianalyte procedures are preferable, particularly if the method is intended for clinical or forensic analysis. Gradient separation was usually applied when the most common antidepressants were included in the methodology however, total chromatographic run times varied widely, from 5 to 40 min [57, 76], depending on column length, extraction technique (offline vs. online techniques), biological matrix or the specific application of the method. 

All these studies are based on spectroscopic techniques. Such determinations are offline, post-reaction analysis. Therefore, sample treatment in the time between reaction and measurement is very important, since it is a source of uncontrollable errors. 

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