Off-line sample preparation

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. 

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. 

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... 

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. 

There has also been a move from slow manual sample preparation techniques to faster automated techniques. Automated sample preparation can be carried out on-line (with sample preparation connected directly to the analysis system) or off-line (sample preparation is automated, but the sample has to be manually transferred to the analysis system). Automated sample preparation offers the potential of performing sample clean-up, concentration, and analyte separation in a closed system. This reduces the sample preparation time, and the whole sample becomes available for analysis, leading to improved limits of detection. It also removes some of the human element from a procedure, thereby improving precision and reproducibility. Eurther-more, automated sample preparation reduces cost by using... 

The sensitivity of the method is evaluated by the limit of detection and quantification. In general, there are no specific criteria for the limit of detection, but for bioanalysis purposes, the limit of quantification is established for the concentration with precision and accuracy lower than 20%. Limits of detection and quantification obtained in CE with UV detection are generally higher than in HPLC, owing to the small optical path in the detection window and injected volumes. As discussed previously, by using preconcentration procedures (off-line sample preparation and/or stacking sample injection techniques) and a more sensitive detection system, particularly LIE and MS detection, suitable detection or quantification limits could be obtained for the apphcation of the methods to the analysis of real samples. 

The higher selectivity attainable due to the use of LC-MS/MS is frequently reduced by decreasing the quality of the sample pretreatment and/or the chromatography. The motivation of this is an increase in the sample throughput. However, this often puts serious demands on the sample pretreatment methods. Also, the off-line sample pretreatment appeared to be the rate-limiting step. Speeding up the sample preparation is one of the most important factors in bioanalysis. One of the most applied approaches is the use of SPE or LLE in a 96-well plate format. Online sample preparation in a 96-well plate format was described by different authors. " Off-line sample preparation seems to be preferred by many researchers. 

The limiting step in achieving fast bioanalysis is off-line sample preparation. This indicates the clear need of online sample pretreatment for speeding up sample preparation. One of most widely applied approaches is the 96-well plate format. While off-Une sample pretreatment seems to be preferred by many researchers, onUne strategies are described as well. " ... 

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