Standards for Quantitative LC-MS Bioanalysis

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. 

Abstract Internal standards play critical roles in ensuring the accuracy of reported concentrations in LC-MS bioanalysis. How do you find an appropriate internal standard so that analyte losses and experimental variations during sample preparation, chromatographic separation, and mass spectrometric detection could be corrected How is the concentration of an internal standard determined Should internal standard responses be monitored during the analysis of incurred samples What are the main causes for internal standard response variations How do they impact the quantitation Why are stable isotope labeled internal standards preferred And yet one should still have an open-mind in their usage for the analysis of incurred samples. All these questions are addressed in this chapter supported by theoretical considerations and practical examples. 

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). 

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]. 

Liquid chromatography coupled to mass spectrometry and tandem mass spectrometry (LC/MS and LC/MS/MS) lead to major breakthroughs in the field of quantitative bioanalysis due to high specificity, sensitivity, speed, and reliability, which becomes the preferred analytical tools for analysis of drugs and metabolites in biological matrices. A simple and rapid method has been developed and validated for the quantitation of vincristine in human plasma by LC/MS/MS with atmospheric pressure chemical ionization using on-line solid-phase extraction. The method uses vinblastine as internal standard and the sample preparation is... 

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