Increasing Sensitivity and Linear Dynamic Range for Drug Quantitation
Using the SCIEX QTRAP® 6500 LC-MS/MS System
Charles River Laboratories continues to invest in the latest analytical technologies, enabling our scientists to provide clients with the most precise and accurate data possible, on time, every time. With the recent installation of three SCIEX QTRAP 6500 System mass spectrometers in our Montreal bioanalytical laboratory, our scientists now have access to the highest level of LC-MS/MS performance for drug quantitation.
Specifications for the QTRAP 6500 system promise up to 5-times greater sensitivity than its predecessor, the QTRAP 5500 system, and up to a 20-fold increase in detector dynamic range, the latter allowing drug quantitation greater than three orders of magnitude. Our scientists can leverage the sensitivity gains afforded by the QTRAP 6500 system for achieving challenging detection limits when sample volume is limited, such as in dried blood spot and capillary microsampling studies, studies involving small rodents or neonatal patients, and matrices such as epithelial lining fluid (ELF), cerebrospinal fluid (CSF) and other tissues. Additional LC-MS/MS sensitivity is crucial when a drug exhibits poor bioavailability, demonstrates low mass spectral ionization efficiency and/or dissociation characteristics, and/or cannot be extracted with high recovery. With optimized scan speeds up to 20,000 Da/sec with polarity-switching speeds of 20 msec, the QTRAP 6500 system is perfectly suited for coupling with our ultra-high performance liquid chromatography (UHPLC) capabilities.
This article presents applications demonstrating the sensitivity and improved dynamic range of the QTRAP6500 system, illustrating the versatility and robustness this platform affords the bioanalytical scientist.
Leveraging the sensitivity gains of the QTRAP 6500 system
Capillary microsampling - Capillary microsampling (CMS) is a technique for collecting small and exact volumes of biological matrices, most commonly blood, plasma, or serum. In rodent toxicology studies, CMS offers the possibility for excluding satellite animals, thereby reducing and refining animal use, maximizing scientific value, increasing productivity and decreasing overall study cost. The low volume collected in CMS approaches makes repeated sampling feasible, and, when coupled with the ability to harvest both plasma and serum from collected whole blood, PK profiles can be generated from three different matrices originating from a single donor. However, low sample volume dictates the development of highly sensitive methodologies, as illustrated in Figure 1, which compares the response from the QTRAP® 5500 and 6500 Systems for an extracted LLOQ of 10 pg/ml (40 fg on-column) for midazolam (MDZ) and hydroxymidazolam (OH-MDZ) from 8 μl of plasma.
Poor ionization efficiency, MRM selectivity, and a 25 pg/ml LLOQ
In this example, our research group was faced with a novel compound that lacked functional groups that could be directly ionized. Parent ions could only be generated indirectly via transient adduction with formate followed by in-source dissociation to furnish [M-H]-. Although ionization was a linear process as a function of concentration in the presence of the formic-acid-containing mobile phase, ionization efficiency was poor in comparison to compounds containing basic or acidic functionalities. While analyte recovery from rat urine was quantitative, selectivity could only be obtained using the least-sensitive MRM transition of two candidate dissociative channels whose response differed 10-fold. As shown in Figure 2, the QTRAP 5500 system failed to meet precision acceptance criteria at the LLOQ (1.25 pg on-column), whereas, the 6-fold increase in signal-to-noise ratio (s/n) afforded by the QTRAP 6500 system readily met regulatory criteria for the same extracted sample set.
Extending linear dynamic range
In a preclinical investigation involving tolerability and toxicity of new formulations of the cancer drug, imatinib, it was necessary to develop an LC-MS/MS plasma method capable of supporting a concentration range spanning four orders of magnitude (1.0–10,000 ng/ml). Previously, such a dynamic range would challenge the saturation limit of the pulse counting detector in the QTRAP 5500 system, resulting in problematic curve fitting. Therefore, ideal concentration ranges were often truncated to practical limitations, with repeats a necessity for samples whose response exceed the upper limit of quantitation (ULOQ). However, with the introduction of the Ion Drive™system technology detector in the QTRAP 6500 system, ultrafast pulse counting (108 cps) with a higher saturation point is feasible, without loss of low-end sensitivity. As outlined in Figure 3, the QTRAP 6500 system demonstrated a linear response for imatinib extracted over four orders of magnitude, without saturation at the ULOQ, thus allowing a complete PK profile without the need for sample repeats (Figure 4). Had the assay been limited to three quantifiable orders of magnitude, ~50% of the toxicology samples collected over the course of the study would have required costly repeat extraction and analysis due to a response greator than ULOQ.
The carryover challenge
With continuing increases in mass spectrometric sensitivity and the ability to quantitate over larger concentration ranges, autosampler carryover becomes a formidable challenge for compounds exhibiting adsorption to materials used in the injection flow path (e.g., needle, needle seal, rotor seal). Carryover can impact the precision and accuracy of an analytical batch (particularly low concentration samples), can lead to inaccurate sample data, and can cause incurred sample reanalysis failure. In regulated bioanalysis, carryover should be ≤ 20% of the LLOQ and is determined by the drug response in a blank sample following a ULOQ injection. Recognizing the importance of eliminating carryover, Charles River Laboratories Montreal has paired the top-performing Shimadzu Sil-30AC autosampler with our Shimadzu Nexera LC-30 AD UHPLC chromatography pumps. Equipped with Pt-coated needle and peristaltic pump, external, internal, and injection port rinses can be configured using a combination of solvents, all controlled by Analyst® Software v1.6 (SCIEX) for GLP compliance. The advanced rinsing capabilities of the Shimadzu Sil-30AC autosampler were recently exemplified in troubleshooting an assay transferred from another laboratory for the determination of methotrexate in plasma (10 μM ULOQ). The provided method required the injection of reagent blank between samples in order to mitigate autosampler carryover. As a first approach to reducing carryover, the extract dilution factor and injection volume were carefully titrated to provide a robust LLOQ response while minimizing the injected on column amount of methotrexate. This resulted in 35% carryover when using an external needle rinse only, a modality reflective of the previous generation of Shimadzu autosampler (Figure 5). However, all carryover could be eliminated when performing a combination of internal needle rinses with three different solutions, an active external needle rinse (wash solution is replaced while the needle remains immersed in the rinse port), and an injection port rinse (Figure 6).
With the sensitivity gains and expanded dynamic range of the SCIEX QTRAP 6500 LC-MS/MS System as a key component in our bioanalytical toolkit, Charles River Laboratories is able to support our customers' most challenging bioanalytical projects, now and into the future. Article prepared by Jeff Plomley and Mohamed Makhloufi. For further information on this topic, please do not hesitate to contact Jeffry Plomley.