Quantitative bioanalysis using LC-MS

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

A wide variety of sample pretreatraent methods are applied in quantitative bioanalysis using LC-MS. The importance of sample pretreatment is evident from the discussion on matrix effects in Ch. 11.5. In general, the three major goals of sample pretreatment are [7] ... 

In order to obtain sufficient accurate results with good precision, an internal standard (IS) must be used in quantitative bioanalysis using LC-MS. Either an isotopically-labelled internal standard (ILIS) or an analogue internal standard (ANIS) can be apphed. 

M. Jemal, Z. Ouyang, Enhanced resolution triple-quadrupole MS for fast quantitative bioanalysis using LC-MS-MS investigations of parameters that affect ruggedness. Rapid Commun. Mass Spectrom., 17 (2003) 24. 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

LC-MS/MS has dramatically changed the way bionalysis is conducted. Accurate and precise quantitation in the pg ml scale is nowadays possible however one has to be aware of certain issues which are specific to mass spectrometric detection such as matrix effects and metabolite crosstalk. With the current growing interest in the analysis of endogenous biomarkers in biological matrices, quantitative bioanalysis with MS has certainly the potential to contribute further in this field with the development of multicomponent assays. Modern triple quadrupole instruments have the feature to use very short dwell times (5-10 ms), allowing the simultaneous determination of more than 100 analytes within the timescale of an HPLC peak. Due to the selectivity of the MS detection the various analytes... 

Quantitative Bioanalysis with High Mass Resolution Prior to the introduction of the API sources for LC-MS, GC-MS was the dominant format for mass spectrometry. Within GC-MS, mass analysis at high resolution using magnetic sector instruments was relatively common, especially in the central mass spectrometry facilities of major corporations and universities. Uses of these instruments included quantitation by GC-HRMS for improved specificity and sensitivity. 

Using a Symbiosis system, Alnouti et al. (2005) and Li et al. (2005) developed online SPE-LC-MS/MS methods for analysis of rat plasma without any prior sample processing. Direct plasma injection resulted in accuracy of 88-111 % and 41 -108% with and without on-line SPE, respectively. The precision was improved from 3-81% without SPE to 0.5-14% with SPE. Furthermore, Alnouti et al. (2005) demonstrated that the cost of quantitative bioanalysis can be reduced by reusing the on-line SPE cartridges up to 20 times without loss of accuracy, precision or analyte recovery. 

Although the scope of application continues to grow, the routine use of LC/MS technologies are now embraced by pharmaceutical researchers. Standard methods that incorporate highly specialized features are routinely developed for a variety of novel applications. Furthermore, many LC/MS applications that deal with quantitative bioanalysis (i.e., pharmacokinetics studies) are frequently outsourced to contract analytical laboratories. Thus, the routine use of LC/MS is a benchmarked commodity for drug development. 

At the time, this application provided a powerful benchmark for the use of LC/MS-based methods in the pharmaceutical industry. This particular assay successfully supported several clinical studies with sensitive and reliable results. This performance was bench-marked on more than 4000 clinical samples and led to a wider scope of application and the development of routine, standard methods for quantitative bioanalysis. 

The use of SRM methods for quantitative bioanalysis represents increased dimensions of mass spectrometry analysis. SRM methods that use APCI-LC/MS/MS for the quantitative analysis of an antipsychotic agent, clozapine, in human plasma were described by Dear and co-workers (Dear et al., 1998). Preclinical development studies of clozapine in rats and dogs used HPLC with fluorescence detection (FLD). With this method, a better limit of quantitation (LOQ) of 1 ng/mL was obtained. As the compound moved into the clinical stages of development, a more sensitive method of analysis was required to obtain rapid metabolic information in support of drug safety evaluation studies. A standard LC/MS/MS method is used for the quantitative analysis of clozapine (I) and four metabolites (II-V) in human plasma (Figure 6.34). 

Internal standards play critical roles in ensuring the accuracy of final reported concentrations in quantitative LC-MS bioanalysis through the correction of variations during sample preparation, LC-separation, and MS detection. The physical-chemical properties of an internal standard, particularly hydrophobicity and ionization properties should be as close as possible to those of the corresponding analyte to better track the variations the analyte experiences. For this reason, stable isotope labeled internal standards should be used whenever possible. However, efforts should still be made to obtain clean extracts, adequate chromatographic separation, and optimized ionization mode and conditions. 

The use of MRM methods for quantitative bioanalysis often reduces sample preparation and analysis time. The MRM method that used LC/ESI-MS/MS for the quantitative analysis of an anticancer drug, Yondelis (Ecteinascidin 743, ET-743, trabectedin. Scheme 9), in human plasma was demonstrated by Rosing et al. [103]. The full-scan mass spectrum of ET-743 (MW 762) contained an abundant [MH+ - H2O] ion at m/z 744 as a result of loss of water molecules from in-source CID (spectrum not shown). The internal standard, ET-729 (Scheme 9, MW 747), exhibited similar performance in the full-scan mass spectrum an abundant [MH+ - H2O] ion at tn/z 730 was produced. The product ion spectra of ET-743 and ET-729 exhibited the most abundant fragment ions at m/z 495 and m/z 479, respectively (spectra not shown). The product ion at m/z 495 (C27H31N2O7) was formed in the collision cell after cleavage of the sulfur bond and ester binding at C-11 [103]. 

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