Capillary UHPLC Coupled with MRM-MS for Quantitative, High-Sensitivity Bioanalysis

Over the past 20 years, liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) has become the primary tool for the bioanalysis of drugs and their metabolites in physiological fluids.1 Advances in genomics, proteomics, combinatorial chemistry, and high-throughput screening have all contributed to an increase in the number of potential drug candidates in the development pipeline of pharmaceutical companies. These advances place a continuous demand on pharmaceutical scientists to develop high-sensitivity, high-throughput, and robust assays for qualitative and quantitative bioanalysis.2 These requirements have also prompted HPLC and MS vendors to make continued improvements to LC-MS instruments, consumables, and methods to help their customers meet the growing demands.

One major area of improvement over the past few years has been the introduction of ultrahigh-performance liquid chromatography (UHPLC) using smaller particle and column technology to provide significant improvements in chromatographic resolution and throughput, with modest gains in sensitivity versus conventional HPLC.3 MS vendors have also improved instrumentation to make bioanalysis more sensitive and robust, reducing the extent of physiological fluid sample cleanup and enhancing overall sample throughput as well. Despite these LC-MS advances, optimum performance is currently achieved using a 2 × 50 mm LC column at flows from 200 to 800 µL/min using conventional electrospray ionization (ESI) sources with MRM-MS on triple quadrupole mass spectrometers. These conditions provide high sample loading capacity (10–1000 µL), robust operation with quantitative results, and high throughput using fast gradient separations in 2–5 min.

As newer drug candidates are developed with higher potency (resulting in lower dosages), the need for improvements in sensitivity for bioanalysis continues to challenge pharmaceutical scientists.4 Since ESI-MS sources are concentration dependent, many attempts have been made to run at lower LC flow rates using smaller i.d. columns with nanospray ionization (NSI) or microspray ionization (MSI) sources in place of a conventional ESI source to improve sensitivity. Nano-LC (100–1000 nL/min) coupled with NSI-MS offers the highest possible sensitivity for LC-MS; however, this technique requires long run times (>30 min inject-to-inject) and significant user intervention to achieve the desired results. Micro-LC (10–100 µL/min) coupled with MSI-MS offers a modest gain in sensitivity over analytical LC- (200–2000 µL/ min) ESI-MS, but the decreased loading capacity makes these gains difficult to realize on physiological fluid samples.

This article describes a nano-capillary UHPLC system coupled with a CaptiveSpray™ ionization- (CSI) MS source (Michrom Bioresources, Auburn, CA) that offers significant gains in sensitivity for bioanalysis without compromising sample throughput, method robustness, or quantitation. The nano-capillary UHPLC provides splitless gradient flows from 0.1 to 10 µL/min at pressures up to 10,000 psi with optimized fluidics so that gradients as fast as 1%/sec can be achieved with minimal extracolumn volume. The system was applied to the determination of buspirone in human plasma samples, and the results show that this technology offers up to a 100-fold sensitivity improvement over conventional LC-ESI-MS with 150-sec inject-to-inject times.



Buspirone and lyophilized human plasma were obtained from Sigma-Aldrich (St. Louis, MO). All solvents were high-purity Burdick & Jackson brand purchased from Honeywell (Muskegon, MI). Acrylate immobilized liquid extraction (ILE) plates were obtained from ILE, Inc. (Ferndale, CA). The Halo™ C18 UHPLC columns used in this work were supplied by Michrom Bioresources.

Immobilized liquid extraction

Figure 1 - Advance nano-capillary UHPLC with CaptiveSpray source on 4000 Q-Trap MS.

Table 1    -    LC-MS conditions used in this study

The lyophilized human plasma was reconstituted with water, and then 200-µL aliquots were diluted with 200 µL of cosolvent (20/80/0.1 ACN/H2O/NH4OH, pH 12) spiked with buspirone (0–200 ng) in wells of a 96-well acrylate ILE plate. After 60 min on a shaker table, the solutions were removed and replaced with 400 µL of extraction solvent (90/10/0.1 ACN/H2O/TFA, pH 2). After shaking for an additional 60 min, 200 µL of the extracts were removed, evaporated to dryness, and reconstituted in 100 µL of analysis solvent (10/90/0.1 ACN/H2O/ TFA, pH 2).

LC-MS conditions

The LC instrumentation and CaptiveSpray source used in this study were from Michrom Bioresources. A 4000 Q-Trap mass spectrometer (AB Sciex, Foster City, CA) was used for all of the experiments, with a standard Turbo-V ESI source (AB Sciex) for analytical runs, a modified Turbo-V ESI source (25-µm-i.d. fused-silica tubing from column to spray tip) for microflow runs, and a CaptiveSpray source for capillary flow runs (Figure 1). LC-MS conditions for each experiment are shown in Table 1