A convenient strategy to overcome interference in LC-MS/MS analysis: Application in a microdose absolute bioavailability study
Introduction
Stable isotopically labeled (SIL) compounds are non-radioactive analogs of the unlabeled compounds, in which one or more atoms are substituted by the corresponding stable isotopes, e.g., 13C, 2H (deuterium or D), 15N and 18O. The physicochemical properties of SIL compounds are very similar, and often considered “identical”, to the unlabeled compounds, which leads to very similar behavior and performance between SIL compounds and their unlabeled version in vitro and in vivo. The most common application of SIL compounds, which utilizes their in vitro similarity, is their use as internal standards (IS) for liquid chromatography–mass spectrometry (LC–MS) based quantitative assays. A SIL-IS performs much better than a structural analog IS on tracking and compensating for the variability of the analyte during sample extraction, chromatographic separation, and mass spectrometric detection. In addition, since it often co-elutes with the analyte or has a very similar retention time, it can also compensate for the matrix effect (ionization suppression or enhancement) caused by the co-eluting matrix components and analytes, therefore, significantly improving the accuracy, precision and robustness of the assay [1]. As a result, SIL-IS is considered the best available and practically the “ideal” IS. The use of SIL-IS is highly recommended for LC–MS based bioanalytical assays, especially for regulated bioanalysis [2,3]. It is worth noting that in some compounds with deuterium labeling the isotope effect of deuterium may slightly change the properties (e.g., lipophilicity) of the compounds [4,5]. This effect is especially true for compounds containing multiple deuterium labels (e.g., 5 or more), in which the property differences between the labeled and unlabeled compound may be substantial enough to cause their partial resolution during chromatographic separation, which could result in their different matrix effects and poor assay quality [6,7]. Thus, when using deuterium labeled compounds as an IS, it is important to evaluate and ensure the isotope effect does not affect the assay quality.
Recently, the application of SIL compounds for in vivo microdose absolute bioavailability (BA) studies has drawn increased attention [[8], [9], [10], [11]]. The traditional approach to assess absolute BA of a drug is to use a crossover design to administer an extravascular (e.g. oral) and intravenous (IV) dose of a drug to the same subjects [12]. The microdose approach allows the concurrent administration of two different routes of a drug to the same subjects: an extravascular dose of the unlabeled drug at therapeutic dose and an IV microdose (100 μg or less) of isotopically labeled drug which serves as a microtracer. This approach avoids the variability between different dosing periods, uses fewer subjects and significantly shortens the clinical study time. The use of an IV micro dose also avoids the need to conduct an animal toxicity study to evaluate the safety of the IV formulation in human. For this approach, an ultrasensitive bioanalytical assay is required to accurately measure the concentration of the microdose drug. Conventionally, accelerator mass spectrometry (AMS), due to its superior sensitivity, is used for the measurement of 14C-labeled drug in microdose absolute BA studies [13,14]. However, the AMS measurement requires chromatographic separation of the 14C-labeled drug from its metabolites to ensure the assay specificity. This could be very challenging, as some metabolites may be difficult to separate from the parent drug, and may not be known or identified during method development. In addition, the unlabeled drug needs to be quantified by a LC–MS/MS assay, which is a different analytical technique, and usually is done in a different lab. This could result in increased data variability and inconsistency, since the samples will be processed by different methods, analyzed by different techniques and conducted in different labs. With the improvements of mass spectrometer instrumentation, LC separation and sample preparation techniques, the sensitivity of LC–MS/MS methods have tremendously improved: they now can often quantify drug concentration in the picomolar or even femtomolar range [15]. This allows the application of LC–MS/MS methods to support microdose absolute BA studies. For the LC–MS/MS approach, SIL drugs are used as the IV microdose drug, which avoid the use of radioactive 14C-labeled drugs in the studies. The SIL drugs used usually are 13C and/or 15N labeled drugs. Deuterium labeled drugs should be avoided if possible to eliminate the potential isotope effect of deuterium, which may alter the pharmacokinetics (PK) of the drug [16]. Another advantage is that LC–MS/MS methods can simultaneously determine the labeled and unlabeled drugs, therefore, significantly improving the data consistency and quality. It can also significantly save cost and time in method development and sample analysis. Because of these notable advantages, the application of LC–MS/MS assays and SIL drugs for microdose absolute BA studies has become the preferred approach if the sensitivity of LC–MS/MS assays can meet the requirements of the studies [14].
To select an appropriate SIL compound as either an IS or an IV dosing microtracer, it is critical to ensure there is no, or very minimal interference between the unlabeled drug and its labeled analog during mass spectrometric analysis. Otherwise, the interference would severely affect the quality and accuracy of LC–MS/MS assays and cause erroneous estimation of the measured concentrations. Especially in microdose absolute BA studies, where the dose of unlabeled drug is much higher than the SIL drug (a minimum of 100-fold higher, sometimes 1000-fold or more), even a very small contribution from the unlabeled drug (estimated based on the same concentration level) will significantly affect the analysis of SIL drug. For example, if the dose of the unlabeled drug is 1000-fold higher than the IV drug, even 0.1% isotopic contribution from the oral drug will cause 100% (0.1%×1000) interference to the IV drug. In addition, since the unlabeled and labeled drug have very similar physicochemical properties, they co-elute during LC–MS/MS analysis. As a result, the high concentration of unlabeled drug may cause severe ion suppression or enhancement to the microdose labeled IV drug [17,18]. To ensure the accuracy and reliability of the LC–MS/MS assay, a differently labeled SIL drug (often with different or additional labeling compared to the IV drug) needs to be used as the SIL-IS to track and compensate for this effect. This results in additional challenge, since a different version of the SIL analog needs to be identified and synthesized, and there should be no or minimal interference from each other among the unlabeled drug, the microdose SIL drug and the SIL-IS during LC–MS/MS analysis.
One major cause of the interference is the isotopic ions of the compounds (isotopic contribution). Due to the presence of natural isotopes for the elements composed of a compound (e.g., 13C for C), there is an isotope distribution for a given compound, e.g., for a compound with a monoisotopic ion of M, there will be ions composed with less abundant isotopes (M + 1, M + 2, M + 3, M + 4 etc.) in its mass spectrum. These isotope ions, depending on their abundance, may cause interference to the analysis of the SIL version of the compound. For example, the M + n ion will interfere with the analysis of the labeled compound with a mass difference of n (e.g., labeled with “n” 13C or other isotopes). The isotope distribution of a compound can be easily calculated using online calculators (e.g. http://www.sisweb.com/mstools/isotope.htm) [11]. Usually, the bigger the number “n” is, the less the isotope distribution of M at M + n, and the less the isotopic contribution (isotopic interference). Hence, increasing the mass difference between the two compounds is the simplest and most common approach to reduce the interference. However, this approach requires the incorporation of more stable isotope labels into the labeled compound, which may significantly increase the difficulty of its synthesis. It may take much longer time and higher cost, and sometimes, may even be impossible to synthesize. The isotope distribution of a compound has been commonly used to estimate the isotopic interference [11]. This approach works well for estimating the interference for MS1 analysis, but often causes overestimation for tandem mass spectrometry analysis (MS2 or MS/MS), since it does not take into account the isotope distribution of fragment ions. Gu et al. [11] developed a new methodology to accurately calculate and predict the isotopic interference in LC–MS/MS assays by using the isotopic abundances of the two fragments (product ion and neutral loss) of the compound. They demonstrated that for the same number and type of labeled atoms, depending on the labeling positions and the selected product ion, the isotopic interference varies significantly. Therefore, by selecting appropriate labeling positions and product ion, they can mitigate the isotopic interference with a minimum number of labeled atoms. However, for this approach, there sometimes may not be a suitable product ion available or it may be difficult to label at the specific positions. In addition, the experimentally determined interference may be more severe than the calculated one due to the presence of impurities of the compounds or from the substances in the biological matrix [8]. These impurities or matrix components often have the same nominal mass as the target compounds, and therefore, cause interference when analyzed by triple quadrupole mass spectrometers with unit resolution. Using high resolution mass spectrometry (HRMS) or monitoring a different product ion can differentiate the interfering compound from the target one, and thus, eliminate the interference [8]. However, this is often limited by the availability of HRMS instrumentation or a different product ion. The sensitivity of the assay may also be much lower when using HRMS or a different product ion.
Indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme which catalyzes the degradation pathway of tryptophan to kynurenine, is a promising immunotherapy target in cancer treatment [19]. Currently, at least seven small molecule IDO1 inhibitors are under clinical development for the treatment of cancer [20]. BMS-986205 (Fig. 1), is a potent and selective IDO1 inhibitor, and is being assessed for safety and efficacy in pivotal clinical studies [21]. To support the microdose absolute BA study of BMS-986205, we needed to select and synthesize two differently labeled versions of the SIL drug to serve as the IV drug and the IS. However, there was interference observed between BMS-986205 and the two originally proposed SIL compounds during LC–MS/MS analysis, which could affect the accuracy and quality of the assay. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the less abundant isotopic ion (instead of the commonly used most abundant monoisotopic ion) in MS analysis. This strategy can minimize the number of stable isotope labels needed to avoid interference, therefore, can greatly reduce the difficulty in synthesizing the SIL compounds and significantly save time and cost. This approach can also be used to reduce the MS response of the analyte, and therefore, avoid the detector saturation. This strategy was successfully applied to the microdose absolute BA study of BMS-986205 in dogs and human clinical study. In addition, applications are presented which utilize this strategy for the selection of SIL-IS.
Section snippets
Chemicals, reagents, materials, and apparatus
BMS-986205 (oral drug), [13C7, 15N]-BMS-986205 (microdose IV drug) and their SIL-IS [13C7, 15N, D3]-BMS-986205 (see structures in Fig. 1) were obtained from Bristol-Myers Squibb (Princeton, NJ, USA). HPLC-grade acetonitrile, ethyl acetate and hexane were obtained from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (> 98%) and acetic acid (HPLC Grade) were purchased from EMD Chemicals (Gibbstown, NJ, USA). Ammonium acetate was obtained from J. T. Baker (Phillipsburg, NJ, USA). Deionized water
Challenges in supporting BMS-986205 microdose absolute BA study
For assessing the absolute BA of a tablet formulation of BMS-986205, a microdosing study using SIL BMS-986205 as the IV component was proposed. The study initially planned to dose the subjects using 200 mg of BMS-986205 for oral dosing and 100 μg of SIL BMS-986205 for concurrent IV microdosing (at Tmax of oral dose). Achieving the desired sensitivity, although not the focus of this paper, is the first challenge and often determines if LC–MS/MS assays can be applied to absolute BA studies. In
Conclusions
A convenient and cost-effective strategy utilizing the isotopic ion was developed to overcome the interference in MS analysis. Using this strategy, only a minimum number of SIL atoms are required for eliminating interference, which results in significant time and cost savings in identifying and synthesizing SIL compounds. This strategy is very useful for the selection of SIL compounds for microdose absolute BA studies and the selection of SIL-IS. It can also be used to reduce the MS response of
Acknowledgements
The authors wish to thank Huidong Gu for helpful discussion and Dr. Mark Rutstein for his review of the manuscript.
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