Method development time

Hyphenated analytical methods usually give rise to iacreased confidence ia results, eaable the handling of more complex samples, improve detectioa limits, and minimi2e method development time. This approach normally results ia iacreased iastmmeatal complexity and cost, iacreased user sophisticatioa, and the need to handle enormous amounts of data. The analytical chemist must, however, remain cogni2ant of the need to use proper analytical procedures ia sample preparatioas to aid ia improved seasitivity and not rely solely on additional iastmmentation to iacrease detection levels. 

The method development time should be minimal, since the process development time is short. 

The use of high flow and fast gradient HPLC has gained a lot of popularity because of the ability to reduce LC/MS/MS cycle times during bioanalysis. In the case of fast gradient HPLC, peak shapes were improved and method development times were minimized, especially when multiple analytes with diverse functionalities had to be separated. Flows as high as 1.5 to 2 mL/min were achieved on a 2.1 x 30 mm Xterra C18 column.7 Details are discussed in a recent review.8... 

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... 

In addition, the use of fast gradients elution mode has become the bioanalytical mainstream as a possible way to improve peak parameters (shape and symmetry) and to minimize method development time, especially for the multi-analytes methods. 

The advantage of generic LC/MS run conditions is that it allows the preparation of an LC/MS separation database that can be referenced for compound mixtures from anywhere in the development and manufacturing process cycle. It trades off resolution for consistency, speed, and a decrease in methods development times. It permits creation of a computer-searchable database of information for all of the compounds being investigated in the company. The mass spectrometer provides sensitivity and resolution gain as well as information on retention times and molecular weights. 

The method of choice is dependent upon the analyte, the assay performance required to meet the intended application, the timeline, and cost-effectiveness. The assay requirements include sensitivity, selectivity, linearity, accuracy, precision, and method robustness. Assay sensitivity in general is in the order of IA > LC-MS/MS > HPLC, while selectivity is IA LC-MS/MS > HPLC. However, IA is an indirect method which measures the binding action instead of relying directly on the physico-chemical properties of the analyte. The IA response versus concentration curve follows a curvilinear relationship, and the results are inherently less precise than for the other two methods with linear concentration-response relationships. The method development time for IA is usually longer than that for LC/MS-MS, mainly because of the time required for the production and characterization of unique antibody reagents. Combinatorial tests to optimize multiple factors in several steps of some IA formats are more complicated, and also result in a longer method refinement time. The nature of IAs versus that of LC-MS/MS methods are compared in Table 6.1. However, once established, IA methods are sensitive, consistent, and very cost-effective for the analysis of large volumes of samples. The more expensive FTMS or TOF-MS methods can be used to complement IA on selectivity confirmation. 

LC/MS/MS to support the clinical development [104-108]. The conventional sample preparation procedures require labor-intensive sohd-phase extraction (SPE) sample pretreatment steps and extensive method development time for the LC/MS drug analysis. One of the popular alternatives to SPE is the resurgence of the on-line SPE or column switching techniques [109-111]. More recently, this strategy has been further developed to determine the drug candidates in human plasma by LC/MS/MS analysis [104,109,112]. 

There are many HPLC methods that are developed during the process of drug development. The chromatographer needs to understand the aim of analysis in order to make judicious choices prior to the commencement of method development and the implications it may have on the final method that is developed. The following should be considered method development time, the maximum run time for analysis, the number of samples expected per week, the complexity of the mixture, the structure of the main analyte (physicochemical properties), possible degradation pathways (i.e., hydrolysis, oxidative, photolysis, dethydration, thermolytic, racemization), and whether the analyte or analytes are ionizable. 

The advantages of on-line automation are the achievement of time savings in relation to the chromatographic method development time. The software can make decisions at any time of the day or night and can immediately communicate this information to the instrument after the completion of the experiment. There is also a more subtle benefit to the link of optimization software to the chromatography data system. Method development wizards with drop-down menus/user-defined fields can simplify the process of configuring the instrument sequence/method prior to a method development session. 

Chiral separations using CE are a popular application 1121 as they are inexpensive, robust and method development time is often rapid. Resolution of chiral basic drugs using cyclodextrins dissolved in a low-pH phosphate buffer has been the most frequently studied application. 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

Method Development Time - A new method based upon GC or HPLC can often be developed in less than one month - particularly for new chemicals belonging to a previously studied class or structural type. IA method developments, on the other hand, may require several months or several years as one synthesizes hapten derivatives, conjugates to protein, immunise. an animal to obtain antibodies, optimizes key assay parameters, and validates the final procedures. This becomes much less of a disadvantage if a stock of antibodies is available for the hapten of interest, in which case method development time for IA can be measured in weeks or a few months. 

The fast-gradient method, in contrast, retains analytes on-column until well after the solvent front has eluted. Overall sample throughput is increased with fast-gradient methods due to reduced analytical run time, decreased method development time, and fewer repeat analyses. Onorato et al. [90] used a multiprobe autosampler for parallel sample injection, short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite at 10 s/sample. Sample preparation employed liquid liquid extraction in the 96-well format. An average run time of 23 s/sample was achieved for human clinical plasma samples. 

Another current trend that is well underway is the use of more specific analytical instrumentation that allows less extensive sample preparation. The development of mass spectrometric techniques, particularly tandem MS linked to a HPLC or flow injection system, has allowed the specific and sensitive analysis of simple extracts of biological samples (68,70-72). A similar HPLC with UV detection would require significantly more extensive sample preparation effort and, importantly, more method development time. Currently, the bulk of the HPLC-MS efforts have been applied to the analysis of drugs and metabolites in biological samples. Kristiansen et al. (73) have also applied flow-injection tandem mass spectrometry to measure sulfonamide antibiotics in meat and blood using a very simple ethyl acetate extraction step. This important technique will surely find many more applications in the future. 

Changing a to 1.05 gives us a 96% reduction in required column efficiency which translates to a 96% reduction in column length, analysis time, solvent consumption, and solvent disposal. Changing selectivity is certainly the most efficient way to improve resolution. However, the cost of changing a is in method development time. 

ELISAs are the most prevalent methods used for detecting antibodies. These assays can be conducted using a variety of approaches, which include direct, indirect, bridging, and competitive inhibition formats (Fig. 8.1). These assay formats offer high throughput, are easily automated, use simple technology, require low capital investment, and enjoy comparatively short method development times. A major... 

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