Bioanalysis quantitative

Liquid chromatography combined with triple-quadrupole mass spectrometers has been used extensively as the analytical method of choice to determine the plasma concentration of compounds. 

With the advent of API sources, LC/MS/MS allows the facile development of quantitative methods that are sensitive, selective, robust, and amenable to the rapid analysis of a majority of small molecules. In order to achieve high-throughput bioanalysis in support of pharmacokinetic studies, many approaches have been reported utilizing automated sample preparation and reducing analysis time by pooling samples, parallel analysis, and fast chromatography. 25,26,152,153 

Before compounds in biological matrices can be analyzed by LC/MS/MS, the samples must undergo a preparation procedure. There are a variety of techniques available for sample preparation including offline sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction) and on-line sample preparation 

SCIEX API 365 equipped with a TurboIonSpray interface operated in positive ion mode. The calibration range 0.124-497 ng/mL was readily validated with a negligible carry-over effect from this system. The method offered a total cycle time of 8 min and completely eliminated the manual sample preparation.  

After the biological samples have been cleaned up, analytes are introduced to LC/MS for analysis. Approaches to shorten the sample analysis time are well documented. Two major strategies 

---

This chapter will review recent advances in mass spectrometry, liquid chromatography, and sample preparation techniques that aim at achieving high throughput. In particular, online solid phase extraction and multiplexed HPLC front ends for quantitative bioanalysis will be discussed in detail. 

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. 

Hsieh S. et al., 2004. Increased throughput of parallel online extraction liquid chromatography /electrospray ionization tandem mass spectrometry system for GLP quantitative bioanalysis in drug development. Rapid Commun Mass Spectrom 18 285. 

Huang M.Q. et al., 2006. Increased productivity in quantitative bioanalysis using a monohth column coupled with high-flow direct-injection hquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 20 1709. 

Jemal M., 2000. High-throughput quantitative bioanalysis by LC/MS/MS. Biomed Chromatogr 14 422. 

Jemal M., Xia Y., and Whigan D.B., 1998. The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples. Rapid Commun Mass Spectrom 12 1389. 

High-Throughput Quantitative Bioanalysis Trends and General Considerations.320... 

High-Throughput Quantitative Bioanalysis in Large Molecule DMPK.333... 

EXPERIMENTAL PERSPECTIVES IN DMPK RELATED HIGH-THROUGHPUT QUANTITATIVE BIOANALYSIS... 

The high-throughput concept for quantitative bioanalysis applies to steps such as assay development, sample collection and sorting, sample preparation, sample analysis, and data processing and reporting. Those processes are closely interlinked and improvement of process throughput is equally important. 

HIGH-THROUGHPUT QUANTITATIVE BIOANALYSIS IN LARGE MOLECULE DMPK... 

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... 

Jemal, M. Ouyang, Z. Enhanced resolution triple-quadrupole mass spectrometry for fast quantitative bioanalysis using liquid chromatography/tandem mass spectrometry investigations of parameters that affect ruggedness. Rapid Commun Mass Spectrom 2003, 17, 24-38. 

Whigan, D. Powell, M. L. Increased throughput in quantitative bioanalysis using parallel-column liquid chromatography with mass spectrometric detection. Rapid Commun Mass Spectrom 2001, 15,... 

Jemal M, Xia YQ. LC-MS development strategies for quantitative bioanalysis. Current Drug Metabolism 7, 491-502, 2006. 

Quantitative Bioanalysis with High Mass Resolution 29... 

Quantitative Bioanalysis with Increased Selectivity Application... 

Over the past two decades, QMF-based quantification assays have become the technique of choice for quantification of drug candidates and their metabolites. Combining a mass spectrometer with LC provides an additional degree of selectivity and makes the combined technique the method of choice for quantitative bioanalysis of drugs and metabolites. Among the mass spectrometer types, QMF are ideal for coupling with LC and atmospheric pressure ionization sources (ESI, APCI, APPI, DART, DESI, etc.) because QMFs have the lowest voltage requirements and vacuum requirements. 

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. 

Figure 1.14. MRM chromatograms of SCH 29851 (383.0. 337.0) and SCH 34117 (311.1 259.1) obtained using Sciex API 3000 (triple-stage quadrupole) and Sciex QSTAR pulsar (Q-TOF). Comparison of MRM chromatograms of SCH 29851 and SCH 34117 obtained at the LOQ (1 ng/mL) using the API 3000 mass spectrometer with those from the Q-TOF mass spectrometer indicated that the S/N ratio is at least 10-20 times better on the API 3000 mass spectrometer. However, the MRM chromatograms from the API 3000 mass spectrometer do not provide the option to further examine the MS/MS spectra whereas the full-scan MS/MS spectra from a Q-TOF based quantitative bioanalysis assay allows one to easily eliminate any questions about false-positive data. (Rephnted with permission from Yang et a ., 2001b.)...

To highlight the advantages of UPLC for quantitative bioanalysis, Yu et al. (2006) enriched rat plasma with alprazolam, ibuprofen, diphenhydramine, naproxen, and prednisolone and compared HPLC-MS/MS and UPLC-MS/MS approaches for quantification of ah five compounds. Apart from the particles that were used to pack the columns, ah other separation and mass spectrometry methods were kept as similar as possible. 

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. 

Table 1.5 compares various fast chromatographic approaches available for LC-MS/MS based quantitative bioanalysis. 

---