Reprinted from American Laboratory e-Supplement March 2010

Technical Article

by Kerry Nugent, Lori Ann Upton, Yixin Zhu, and Chris Loran

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 high­sensitivity, 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 ultrahighperformance 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 100fold sensitivity improvement over conventional LC-ESI-MS with 150sec inject-to-inject times.

Experimental Materials Buspirone and lyophilized human plasma were obtained from SigmaAldrich (St. Louis, MO). All solvents were high-purity Burdick & Jackson brand purchased from ­Honeywell (Muskegon, MI). Acrylate immobilized liquid extraction

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

(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 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 ­C a p t i v e S p r a y s o u r c e u s e d i n this study were from Michrom ­B ioresources. 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.

Results and discussion HPLC conditions A universal fast LC gradient was used so that this methodology could also be applied to the wide variety of potential drug candidates and their metabolites encountered in ADMET (absorption, distribution, metabolism, excretion, and toxicity) and DMPK (drug metabolism and pharmacokinetics) bioanalyses, which often cover a wide range of polarities. The 2.7-µm, 90-Å Halo C18 material was chosen because it offers UHPLC resolution and speed at conventional HPLC column pressures; thus the methodology can be applied to HPLC, UHPLC, or UPLC ® (Waters Corp., Milford, MA) instrumentation. Each LC system was plumbed to optimize

Table 1 LC-MS conditions used in this study Parameter LC system LC autosampler LC column Flow (pressure) Linear velocity Solvent A Solvent B Gradient Total run time MS system MS source Buspirone MRM Spray voltage Source temp. Gas flows

Analytical flow runs Paradigm MS2-MA Advance Bio-Cool AS 2 × 50 mm Halo C18 500 µL/min (3350 psi) 2.5 mm/sec 0.1% HCOOH in H2O Acetonitrile 10-90%B in 90 sec 2.5 min 4000 Q-Trap Turbo-V ESI 386.3 → 122.2 + 4500 350 ºC 10/20/20

Microflow runs Paradigm MS2-NC Advance Bio-Cool AS 0.5 × 50 mm Halo C18 32 µL/min (3500 psi) 2.5 mm/sec 0.1% HCOOH in H2O Acetonitrile 10–90%B in 90 sec 2.5 min 4000 Q-Trap µ Turbo-V ESI 386.3 → 122.2 + 4500 350 ºC 10/20/20

Capillary flow runs Advance UHPLC Advance Bio-Cool AS 0.2 × 50 mm Halo C18 5 µL/min (3450 psi) 2.5 mm/sec 0.1% HCOOH in H2O Acetonitrile 10–90%B in 90 sec 2.5 min 4000 Q-Trap Advance CSI 386.3 → 122.2 + 1400 N.A. 0/0/0

a

umn run at 500 µL/min. The results of the microflow runs showed that the gain in sensitivity for micro-LCMSI-MS was only 5× versus the LCESI-MS runs, which was attributed to the fact that the Turbo-V source is designed for flows from 200 to 800 µL/min, limiting the gains achievable simply by lowering the flow rate.

b

Figure 2 a) ESI-MS of 1000-pg buspirone standard with sensitivity of 1.3e5. b) ESI-MS of 10 pg buspirone in human plasma with sensitivity of 1.4e3.

Figure 3 Capillary ESI-MS of 100 pg buspirone standard with sensitivity of 6.2e4.

performance at the three flow rates used in this study, resulting in similar peak widths and retention times to allow direct comparison of sensitivity across the flow range.

Analytical flow runs The initial runs were done using conventional LC-ESI-MS, similar to those currently employed by many pharmaceutical scientists for ADMET and DMPK bioanalyses. Figure 2a shows a representative LC-MS trace for a 100-µL injection of a 10-ng/mL buspirone standard (1000 pg); Figure 2b is a representative LC-MS trace for a 100-µL injection of 0.1 ng/mL buspirone in human plasma extract (10 pg) using an analytical flow rate of 500 µL/min with associated conditions listed in

Table 1. These results demonstrate that the conventional analytical flow conditions used for ADMET and DMPK bioanalysis give good linearity, with a lower limit of detection (LLOD) of 1 pg buspirone and a lower limit of quantitation (LLOQ) of 2.5 pg buspirone. In order to achieve a concentration LLOQ of 25 pg/ mL buspirone in plasma, a 100µL injection of human plasma extract was required.

Microflow runs

Capillary flow runs The final set of runs was performed using capillary LC-CSI-MS to see if the Advance splitless nano­capillary UHPLC system coupled with a CaptiveSpray source would deliver the desired sensitivity gains of a low flow system without impacting the sample throughput, method robustness, quantitative accuracy, or precision typically achieved with LC-ESI-MS. Figure 4 shows a representative LC-MS trace for a 1-µL injection of a 10-ng/mL buspirone standard (10 pg) using the capillary flow run conditions from Table 1. To achieve the same linear velocity used for the analytical flow runs, the capillary flow runs utilized a 0.2 × 50 mm column run at 5 µL/ min. This was expected to give a theoretical increase in sensitivity of 100× versus the analytical runs on the 2.0 × 50 mm column run at 500

The second set of runs was done using micro-LC-MSI-MS to determine if the sensitivity could be improved by scaling down the flow and minimizing system volumes, while still using the Turbo-V ESI source on the 4000 Q-Trap MS. Figure 3 shows a representative LC-MS trace for a 10-µL injection of a 10-ng/mL buspirone standard (100 pg) using the microflow run conditions from Table 1. To achieve the same linear velocity used for the analytical flow runs, the microflow runs utilized a 0.5 × 50 mm column run at 32 µL/min, which was expected to give a theoretical increase in sensitivity of 16× versus the analyti- Figure 4 CSI-MS of 10 pg buspirone standard cal runs on the 2.0 × 50 mm col- with sensitivity of 1.4e5.

mL. Recoveries for the buspirone spiked into human plasma using the ILE sample cleanup and capillary LC-CSI-MS method ranged from 84 to 98%, with CVs from 5 to 12% (n = 5).

MRM-MS response for buspirone did not change significantly over the duration of this study (retention time CV for 4032 runs was 0.27%, and area CV for 4032 runs was 12.8%). The 4000 Q-Trap MS was visually inspected and cleaned before and after the robustness experiment, and the Q0 region of the MS appeared to be at least as clean as it was after a similar number of plasma extract samples were run on the conventional LC-ESIMS system.

Capillary flow sensitivity Figure 5 curve.

CSI-MS buspirone standard calibration

µL/min as the relative concentration on the column is increased as a square of the radius. The results of the capillary flow runs showed that the theoretical 100× gain in sensitivity for buspirone was indeed achieved using the capillary LCCSI-MS versus the conventional LC-ESI-MS method.

Conclusion Capillary UHPLC coupled with CaptiveSpray ionization-MRM-MS offers a highly sensitive alternative to conventional LC electrospray ionization-MRM-MS for bioanalysis. With 100× higher sensitivity, the technique is well suited for applications that are sample limited, including newborn screening,5 dried bloodspot analyses, 6 small animal ADMET studies,7 and microdosing studies in preclinical and early-phase clinical trials.8 The Advance splitless nano/capillary UHPLC introduced in this study delivered highresolution separations at nano- to capillary flows with minimal extracolumn volume, providing high-

Capillary LC-CSI-MS method robustness

Capillary flow quantitative accuracy and precision

The robustness of the method was assessed by running spiked plasma extracts (1 pg buspirone in 1 µL of human plasma extract) 24 hours per day for one week (4032 runs with a 150-sec inject-to-inject cycle time). Although the column backpressure increased by about 10% over the course of the runs, the peak shape, retention time, and

Using the capillary LC-CSI-MS method, a calibration curve for buspirone was constructed (Figure 5), which showed good linearity over the 104 dynamic range. This calibration curve was then used to analyze a variety of 1-µL aliquots of human plasma extract spiked with buspirone at levels from 0.1 to 1000 ng/

a

Figure 6a shows a representative LC-MS trace for a 1-µL injection of 10 ng/mL buspirone in human plasma extract (91% recovery). Figure 6b shows a representative LC-MS trace for a 1-µL injection of 0.01 ng/mL buspirone (10 fg) in human plasma extract (LLOD), and Figure 6c shows a representative LC-MS trace for a 1-µL injection of a human plasma extract blank.

b

c

Figure 6 a) CSI-MS of 10 pg buspirone in human plasma with sensitivity of 1.3e5. b) CSI-MS of 10 fg buspirone in human plasma with sensitivity of 1.3e2. c) CSI-MS of human plasma blank.

throughput gradient separations comparable to analytical UHPLC, while using 100-fold less sample and solvent. The efficiency generated from the Halo C18 column particles provided UHPLC separations at conventional HPLC pressures, with the analytical robustness necessary for ADMET and DMPK assays. The CaptiveSpray source utilized for this work was designed to provide the sensitivity of nanospray with the robustness of electrospray and, when coupled with the Advance UHPLC system and the 4000 Q-Trap MS, delivered a 100× improvement in sensitivity compared with conventional LC-ESI-MS. Future plans include applying this LC-CSI-MS method to a broad range of potential drug candidates, coupling the Advance UHPLC and CSI source to

other triple quadrupole MS systems to further validate the methodology, and expanding system suitability investigations to validate that the methodology meets the requirements for qualitative and quantitative bioanalysis.9

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5. Griffiths, W.J.; Wang, Y.; Karu, K.; Samuel, E.; McDonnell, S.; Hornshaw, M.; Shackleton, C. Clin. Chem. 2008, 54(8), 1317–24. 6. Spooner, N.; Lad, R.; Barfield, M. Anal. Chem. 2009, 81(4), 1557–63. 7. Herman, J.L. Int. J. Mass Spectrom. 2004, 238(2), 107–17. 8. Ni, J.; Ouyang, H.; Aiello, M.; Seto, C.; Borbridge, L.; Sakuma, T.; Ellis, R.; Welty, D.; Acheampong, A. Pharm. Res. 2008, 25(7), 1572–82. 9. Briscoe, C.J.; Stiles, M.R.; Hage, D.S. J. Pharm. Biomed. Anal. 2007, 44(2), 484–91.

The authors are with Michrom ­Bioresources, 1945 Industrial Dr., Auburn, CA 95603, U.S.A.; tel.: 530-888-6498; fax: 530-8888295; e-mail: [email protected].