Bioanalysis method development

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... 

Chovan et al.30 described a system that integrates different components of bioanalysis including automatic in vitro incubation, automatic method development (mainly SRM transitions for LC/MS/ MS analysis), and a generic LC method for sample analysis to minimize human intervention and streamline information flow. Automaton software (Applied Biosystems) was used for automatic MS method development. Flow injection was used instead of a HPLC column to decrease run time to 0.8 min per injection. Two injections were performed. The first was performed to locate the precursor ion and optimal declustering potential (DP). The second injection was performed to locate the product ion and optimal collision energy (CE). 

A systematical approach of sample preparation methods and optimisation of the quality aspects of sample preparation may enhance the efficiency of total analytical methods. This approach may also enhance the quality and knowledge of the methods developed, which actually enhances the quality of individual sample analyses. Unfortunately, in bioanalysis, systematical optimisation of sample preparation procedures is not common practice. Attention to systematical optimisation of assay methods has always been mainly on instrumental analyses problems, such as minimising detection limits and maximising resolution in HPLC. Optimisation of sample extraction has often been performed intuitively by trial and error. Only a few publications deal with systematical optimisation of liquid-liquid extraction of drugs from biological fluids [3,4,5]. 

Foundation A platform of performance established Method development/rehnement analysis benchmarks quantitative bioanalysis methods established new product development. 

Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS... 

Cerivastatin is administered in its acid form. It forms seven acid and lactone biotransformation products. The simultaneous bioanalysis of all eight components in human scram was reported using [Djl-ILIS for cerivastatin and its lactone [52]. The method was similar to the method developed for atorvastatin [51], described above. The LOQ was 0.01 ng/ml for cerivastatin and its lactone, and between 0.05 and 0.5 ng/ml for the other biotransformation products. The method was validated. 

In the bioanalysis of finasteride in human plasma [69], the method development was initially directed at the use of ESI-MS. An ANIS was used. While a precision in the finasteride peak area better than 5%RSD was obtained upon injection of standard solutions, precision values ranging between 17 and 43%RSD were obtained with extracts of five different sources of control human plasma. The matrix effects were further evaluated in two ways. The response for the ANIS in post-extraction spiked plasma samples was evaluated for the five different plasma extracts. In addition, the effect of enhancing the selectivity of the extraction and the efficiency of the LC separation was evaluated. Although these measures showed some improvement, the precision and accuracy of the ESI-MS method remained inadequate to support clinical studies. Therefore, an LC-MS method based on the use of APCI was successfully developed and validated. 

The most adequate method to eliminate matrix effects is the use of an ILIS. An ILIS shows (almost) identical behaviour to the target analyte in both sample pretreatment, chromatography, and analyte ionization. The data reported in the bioanalysis of mevalonic acid [23] indicate that this issue needs proper attention during method development and validation (Ch. 11.3.4). 

DT Rossi. Automating solid-phase extraction method-development for biological fluids trends and applications in bioanalysis, LC-GC 17 S4—S8, 1999. 

Another distinction about discovery bioanalysis is that ease of methods development is often more important than the net speed at which samples can be processed. This statement is again a manifestation of the high number of NCEs that are encountered in discovery. Consequently, enormous growth has occurred with on-line methods that combine sample preparation and analysis in a single injection format. Although several formats exist, the common link to all on-line methods is that they invoke column-switching techniques. The popularity of these methods can be traced, in part, to the ability to adjust extraction/analysis conditions on-the-fly and leads to extremely facile method development. In the section that follows, off-line and on-line methods are considered separately. Coverage of these subtopics can also be found in a number of review articles [4—7,44,45]. 

In our laboratory, we rely heavily on 96-well protein precipitation as the primary form of sample preparation. Not only have we found this approach to be the mostuniversal, butitis also the most cost-effective method for discovery bioanalysis. To overcome limitations associated with protein precipitation, CS methods were developed (Figure 11.3) to permit on-line sample cleanup [49]. In addition, fast gradient elution has been used with this strategy to allow expedient method development and fast sample analysis. 

The field of bioanalysis remains dominated by HPLC, despite the fact that several other chromatographic forms have been interfaced to MS, which include gas chromatography, supercritical fluid chromatography, and capillary electrophoresis. The popularity of RP-LC stems from its instrumental simplicity, wide scope of application, and relative ease of method development. This section primarily focuses on RP-LC, with attention also given to the recent resurgence in normal-phase methods (NP-LC). 

The distinctions within the drug-discovery environment previously noted naturally favor the use of simpler and faster sample-preparation techniques. However, most all sample-preparation methods are potentially viable choices, as recent applications have demonstrated method-development techniques that are both rapid and selective. Also, there is value for the quick development of a bioanalytical method with selectivity for a series of similarly related structures, as that one method can often meet the needs for PK and toxicokinetic (TK) studies well into the discovery process, rather than just for one initial study. The variety of sample-preparation methodologies for bioanalysis are now detailed. 

Because LBAs are conceptually simple and operationally easy to perform, there is often a perception among scientists that LBA method development and validation should be fairly straightforward and simple to conduct. In reality, establishment of LBAs for GLP-compliant bioanalysis to support PK/TK assessments of biotherapeutics is often a challenging task. Successful LBA establishment involves a systematic... 

With the increasing number, diversity, and complexity of compounds being analyzed, UPLC presents the possibility to extend and expand the utility of separation science. Today, UPLC is widely used for metabolite identification analysis of natural products and herbal medicines pharmacokinetic, toxicity, degradation, bioanalysis, and bioequivalence studies quality control and in drug discovery determination of pesticides and separation of various pharmaceutical-related small organic molecules, proteins, and peptides. UPLC is also used for impurity profiling, method development, and validation performed in quality assurance and quality control laboratories [46,47,56-69]. 

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