Abstract
Aim:
We developed a generic high-performance liquid chromatography mass spectrometry approach for quantitation of small molecule compounds without availability of isotopically labelled standard.
Methods:
The assay utilized 50 μL of plasma and offers 8 potential internal standards (IS): acetaminophen, veliparib, busulfan, neratinib, erlotinib, abiraterone, bicalutamide, and paclitaxel. Preparation consisted of acetonitrile protein precipitation and aqueous dilution in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 μm, 2 mm × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. Performance of the generic approach was evaluated with seven drugs (LMP744, olaparib, cabozantinib, triapine, ixabepilone, berzosertib, eribulin) for which validated assays were available.
Results:
The 8 IS covered a range of polarity, size, and ionization; eluted over the range of chromatographic retention times; were quantitatively extracted; and suffered limited matrix effects. The generic approach proved to be linear for test drugs evaluated over at least 3 orders of magnitude starting at 1–10 ng/mL, with extension of assay ranges with analyte isotopologue MRM channels. At a bias of less than 16% and precision within 15%, the assay performance was acceptable.
Conclusion:
The generic approach has become a useful tool to further define the pharmacology of drugs studied in our laboratory and may be utilized as described, or as starting point to develop drug-specific assays with more extensive performance characterization.
Keywords: Generic assay, tandem mass spectrometry, validation
Graphical Abstract
1. Introduction
Early clinical trials are commonly supported by quantitative assays to define the pharmacokinetics of the therapeutic agent under investigation, often including metabolites. These assays will generate data that may support submissions to regulatory authorities, and are therefore properly validated, e.g. following the U.S. Food and Drug Administration (FDA) requirements [1–13]. These efforts are resource intensive and often include the synthesis of isotopically labeled standards (ILS) to improve the precision and robustness of the data generated. However, there are many instances where a project does not require full assay validation, but rather a fit-for-purpose approach, which would not justify generating ILS but rather the use of an unrelated internal standard (IS), and a lower bar for validation efforts. This would be appropriate early in the development of a compound or drug candidate. Although some organizations, and many larger pharmaceutical companies appear to have generic assays with a fixed unrelated internal standard for semi-quantitative screening-type pharmacokinetic needs [14, 15], there is a lack of detailed publications on easy-to-implement generic fit-for-purpose approach assays and their performance relative to appropriately validated assays. E.g. Xu et al. described a drug discovery type generic assay, but did not elaborate how this approach was developed, and only evaluated its performance with a single, unknown compound [15]. Consequently, it is unclear what level of confidence is associated with data generated with this type of assay. Even if the associated data are used to inform decisions where lower assay performance is acceptable, e.g. rank ordering a group of analogues based on achieved plasma exposure, this lack of assay performance represents a knowledge gap.
The goal of this study was to develop a generic, fit-for-purpose liquid chromatography mass spectrometry approach for quantitation of small molecule drug compounds, and evaluate their performance against fully validated assays.
2. Methods
2.1. Chemicals and reagents
Paclitaxel (>99.5%), olaparib (>99%), cabozantinib (>99%), and erlotinib (>99%) were purchased from LC Labs, (Woburn, MA 01801). Acetaminophen (USP), triapine (99.4%), and busulfan (100.7%) were purchased Sigma-Aldrich Co., (St Louis, MO 63178). Abiraterone (98%), neratinib (>99%) and bicalutamide (98%) were purchased from Toronto Research Chemicals, (Ontario, Canada, M3J 2K8). Veliparib (99.7%) was provided by AbbVie. Ixabepilone (99.4%) was provided by Bristol Meyers Squib. LMP744 and eribulin (both clinical grade) were provided by the National Cancer institute, (Bethesda, MD 20892). Berzosertib (M6620, VX-970, 99.5%) was provided by Vertex Pharmaceuticals, (Boston, MA 02210). Acetonitrile, methanol, and water (all HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Formic acid (reagent grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Control EDTA and heparin anticoagulated human plasma was purchased from Lampire biological Laboratories, Inc (Pipersville, PA 18947). Nitrogen for evaporation of samples was purchased from Valley National Gases, Inc. (Pittsburgh, PA, USA). Nitrogen for mass spectrometric applications was purified with a Nitrogen Generator (Parker Balston, Haverhill, MA, USA).
2.2. Chromatography
The LC system consisted of an Agilent (Palo Alto, CA, USA) 1200 SL autosampler and binary pump, a Phenomenex (Torrance, CA USA) Kinetex C18 100Å (2.6 μm, 50 × 2 mm) column, and a gradient mobile phase with a gradient delay volume of 320 μL, pumped at 0.4 mL/min. Mobile phase solvent A was 0.1 % (v/v) formic acid in water, and mobile phase solvent B was 0.1% (v/v) formic acid in acetonitrile. The initial mobile phase composition was 5% solvent B. The percentage of B was increased linearly to 95% over 5.0 min and held at 95% B from 5 to 6 min. At 6.1 min, the percentage of solvent B was decreased back to 5%, and kept at 5% until 8 min, followed by injection of the next sample.
2.3. Mass spectrometry
Mass spectrometric detection was carried out using an AB SCIEX (Concord, ON, Canada) 4000 quadrupole hybrid linear ion trap tandem mass spectrometer (4000QTRAP) with electrospray ionization in positive multiple reaction monitoring (MRM) mode. The settings of the mass spectrometer for all analytes were as follows: curtain gas 40, collisionally activated dissociation (CAD) gas 10 Ion transfer voltage 5000 V, probe temperature 500°C, ion source gas 1 (GS1) 40, ion source gas 2 (GS2) 40, entrance potential 5 V, collision cell exit potential 15 V, and dwell time 0.1 s. The declustering potential, collision voltage and MRM m/z transitions monitored are listed in Suppl.Table 1.
2.4. Preparation of standards and samples
2.4.1. Internal standard working solution
Stock solutions were prepared separately at 1 mg/mL for all IS and stored at −80 °C. Busulfan, neratinib, erlotinib and veliparib were dissolved in acetonitrile. Acetaminophen was dissolved in methanol while bicalutamide, paclitaxel, and abiraterone were dissolved in DMSO. The compounds were then combined in acetonitrile to obtain the following concentrations (ng/mL): busulfan 1000, veliparib 10, erlotinib 5, abiraterone 500, paclitaxel 400, neratinib 400, bicalutamide 400, and acetaminophen 400. An aliquot of 150 μL of this IS working solution was added to each plasma sample.
2.4.2. Test drugs
Each of the following were dissolved separately to obtain a 1 mg/mL solution and were stored at – 80 °C: olaparib was dissolved in DMSO:methanol (20:80); Berzosertib, triapine and cabozantinib were dissolved in DMSO; ixabepilone and eribulin were dissolved in acetonitrile; LMP744 was dissolved in water with 0.1% formic acid. Stock solutions were verified by duplicate independent preparation from powder.
2.4.3. Calibration and quality control samples
On the day of assay the respective test drug or analyte was diluted stepwise with acetonitrile to obtain 100, 10, 1 and 0.1 μg/mL solutions. In a final step, these calibration working solutions were diluted in human plasma to produce the following plasma concentrations: 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 ng/mL in 500 μL aliquots. Quality control (QC) samples were prepared in human plasma at 2, 20, 125, 800, and 8000 ng/mL. The final concentration of acetonitrile was 5% or less for each calibration or QC level.
2.4.4. Sample preparation
A volume of 50 μL of the standard, QC, or sample plasma was pipetted into a well of a 96 well plate (Agilent 31 mm deep 96 well plate, part number 5042–6454) and 150 μL of IS mix (section 2.4.1) was added to each well. The well plate was vortexed for 1 min on a Vortex Genie-2 set at 4 (Model G-560 Scientific Industries, Bohemia, NY, USA). The well plate was then centrifuged (Model 5810 R Eppendorf, Hauppauge, NY, USA) at 2,500 × g at room temperature for 10 min. A volume of 125 μL of the resultant supernatant was transferred with an 8-channel pipette to a clean well plate (Agilent 31 mm deep 96 well plate, part number 5042–6454) and 500 μL of water was added, followed by vortexing for 10 seconds on a Vortex Genie-2 set at 4. The well plate was then placed in the autosampler and 5 μL was injected in the LC-MS/MS system.
2.5. Assay development
2.5.1. Sample preparation
While we aimed for a 96-well format, we did include a microfuge sample preparation as comparator. In addition to the final protein precipitation method described above (2.4.4) which used the Agilent 31 mm deep well plate, five other 96 well extraction plates were evaluated. A Supelco protein filter plate was evaluated first, but the requirement of a vacuum apparatus and dry down equipment for 96-well format prompted us to include simpler approaches also. Acetonitrile was chosen over methanol as the organic solvent for protein precipitations thanks to its greater ability to precipitate proteins and create a cleaner final matrix [16].
Ten microliters of acetonitrile IS mix were added to 50 μL of plasma sample before sample was added to the plates. The concentrations in the IS mix was as follows: busulfan, 2000 ng/mL; veliparib, 20 ng/mL; erlotinib, 10 ng/mL; abiraterone, 100 ng/mL; paclitaxel, 800 ng/mL; neratinib, 800 ng/mL; bicalutamide, 800 ng/mL; and acetaminophen, 800 ng/mL. The plasma samples were processed for each well plate according to the manufacturer’s instructions. A Vortex Genie was used for vortexing, a Waters extraction plate manifold (SKU: 186001831) was used for sample elution, and an Organomation (Model 20406, Berlin, MA, USA) 96 well evaporator was used to dry samples under nitrogen at 37 °C. All extracts were ultimately eluted into an Agilent 31 mm 96 well deep plate under (5 mm Hg) vacuum, followed by drying down. Ten microliters of acetonitrile were added followed by vortexing, addition of 40 μL of water, further vortexing, and injection of 5 μL. For additional detail on each of the sample preparation approaches evaluated, see Supplement.
2.5.2. Extraction recovery and matrix effect
We determined the extraction recoveries of all IS from plasma by comparing the absolute response of an extract of control plasma to which these IS had been added after protein precipitation, with the absolute response of an extract of plasma to which the same amounts had been added before protein precipitation. The ion-suppression by plasma matrix components was defined as the decrease of the absolute response of an extract of control plasma to which IS had been added after the protein precipitation relative to the absolute response of reconstitution solvent to which the same amount of each respective IS had been added. Experiments were performed at two QC concentrations, in triplicate.
2.5.3. Selectivity and specificity
To investigate whether endogenous matrix constituents interfered with the assay, six individual batches of control, drug-free human plasma were processed and analyzed according to the described procedure (section 2.4.3). Responses of IS at the working solution concentrations (section 2.4.1) were compared with the response of the blank samples.
2.5.4. Stability
We determined the bench top stability of the IS working solution at room temperature for 6 hours and the freeze thaw stability of the IS working solution for 3 cycles. In both experiments 150 μL of IS working solution was added to 50 μL of water in a 96 well plate. The plate was briefly vortexed and then 500 μL of water was added followed by vortexing and 5 μL was injected into the LC-MS/MS system. The bench top stability was assessed by comparing the areas of the 8 IS in the IS working solution (n=4) that was freshly made to the areas of the same IS of the same solution which was allowed to stand at room temperature for 6 hrs (n=4). The freeze thaw stability was assessed by comparing the areas of the 8 IS from a freshly made working solution (n=4) to the same solution that was frozen at −80 °C and thawed and refrozen once a day for three consecutive days. After the third day the IS working solution was thawed and analysed (n=4).
2.5.5. IS signal stability
We determined the effect of multiple injections over time on the IS counts. This was done by processing (see section 2.4.3) and analysing a 96 well plate of control plasma with IS.
2.6. Performance evaluation
We tested the applicability of this newly developed generic approach assay by comparing its performance on seven test drug analytes for which we had access to assays that were previously validated, often to FDA guidance [6–8, 17–20], see also Suppl.Table 2. Most of these validated assays used an ILS. Tuning, QC sample preparation, and data analysis were performed as described in the Supplement. Residuals of the generic approach calibrator and QC samples were calculated. Cross-validation consisted of analysing QCs from the established assay with the established validated method (N=6) and with the generic approach assay (N=6) for comparison of respective back-calculated concentrations, bias, and precision metrics. Matrix effects for these test analytes were determined (N=4) to further characterize this behaviour in our assay.
2.7. Application of the assay
In support of a rogaratinib murine xenograft PK-PD study conducted by the Division of Cancer Treatment and Diagnosis of the National Cancer Institute plasma and tumor (homogenized in 3 parts PBS (v/g)) were analyzed using this generic assay in plasma. To our knowledge, a validated assay for rogaratinib has yet to be described in the literature. Calibrators in tumor homogenate were able to quantitate plasma QCs at 150, 1,500, and 15,000 ng/mL (N=4) with adequate accuracy (86.3–95.2%) and precision (<7.6%), with IS and rogaratinib experiencing a −11.5% and −4.9% tumor relative to plasma matrix effect. Rogaratinib was dosed orally at 27, 55, or 164 mg/kg to NCI-H716 tumor-bearing female athymic nude (nu/nu NCr) mice. Terminal samples were obtained at 1, 2, 4, 8, and 24 h after dosing. Pharmacokinetic parameters were calculated non-compartmentally using PK Solutions 2.0 (Summit Research Services, Montrose, CO http://www.summitPK.com).
3. Results and Discussion
3.1. Assay development
Our goal was to develop a generic analytical approach by which it would be possible to quantitate compounds sent for study that did not require a full validation to FDA guidance and for which a stable ILS is unavailable. After selection of IS (see below), their analytical behaviour was then utilized to guide further method development. It was our objective to achieve a chromatographic run that would be able to elute the internal standards in the shortest time possible, and for minimal sample preparation. A 96 well plate format was adopted, which requires less sample preparation than using microcentrifuge tubes, glass tubes and HPLC vials, and also because this allows for automation if desired.
3.1.1. Chromatography
It was our objective to achieve a chromatographic run that would be able to elute potential unknown compounds in a reasonable amount of time i.e. less than 10 minutes. To meet this goal, 6 different columns and different HPLC gradients were evaluated using acetonitrile and water with formic acid. The columns tested were: Phenomenex Synergi Hydro-RP (50 × 2 mm 4 μm), Phenomenex Luna Phenyl-Hexyl (50 × 2 mm 3 μm), Phenomenex Polar RP (100 × 12 mm 4 μm), Phenomenex Polar RP (50 × 2 mm 4 μm), Phenomenex Kinetex (50 × 2 mm 2.6 μm) and Shodex (Showa, New York, NY, USA) ODP2 HP-2B (150 × 2 mm). The Phenomenex Kinetex column with a linear gradient of 5% acetonitrile to 95% in 5 minutes allowed for evenly spread out retention times and adequate peak shapes of the 16 candidate IS. Acetonitrile was chosen over methanol by virtue of the lower HPLC pump back pressure which allows a higher flow rate during column re-equilibration and a shorter run time.
3.1.2. IS selection
The following 16 compounds were evaluated: abiraterone, acetaminophen, belinostat, bicalutamide, busulfan, enzalutamide, erlotinib, etoposide, 5-fluoro-2’-deoxycytidine, imatinib, isofludelone, ixabepilone, mefenamic acid, neratinib, paclitaxel, and veliparib. Out of these 16 compounds the following eight compounds were selected for the IS mix based on retention time and peak shape: abiraterone, veliparib, paclitaxel, erlotinib, neratinib, bicalutamide, busulfan and acetaminophen. In this selection, consideration was given to representation of small size (acetaminophen), large size (paclitaxel), polar (veliparib), non-polar (abiraterone), acid (acetaminophen), and base (neratinib) characteristics. The elution pattern of this selection of IS candidates is shown in Fig. 1.
Fig. 1.
Elution pattern of IS selected for inclusion in the generic assay. In order of elution: acetaminophen (1.2 min), veliparib (1.8 min), busulfan (2.5 min), neratinib (3 min), erlotinib (3.6 min), abiraterone (4.4 min), bicalutamide (4.8 min), and paclitaxel (5.1 min).
3.1.3. Mass spectrometry
We aimed to be able to quantitate from 1 to 10,000 ng/mL covering potentially low and high relevant concentration ranges. Based on experience and our desire to follow a dilute-and-shoot approach, concentrations lower than 1 ng/mL were considered not to be practically achievable. While our detector is expected to be sensitive enough for the 1 ng/mL lower limit for some agents, commonly linearity is lost at a dynamic range of about 3 orders of magnitude or more. To maintain linearity at the higher concentration range, the mass spectrometric signal can be diluted by monitoring successive isotopologues. This approach may achieve linearity at higher concentrations at the expense of the lower limit of quantitation using that isotopologue. Our approach was to select the isotopologues of one AMU and two AMU higher than the conventional [M+H]+ ion for both the precursor and the product ion of each transition. This allows for the possibility of using the conventional [M+H]+ transition for the lower concentration range (1–300 or 1–1000 ng/mL) and the isotopologue MRM channels monitoring the m/z values of the respective 13C1- and 13C2-isotopologues for higher concentration ranges, avoiding signal saturation. Just considering the first mass selection, monitoring the 13C1-isotopologue of a molecule with ten or 20 carbons, amounts to a signal dilution by approximately a factor of 8.5 and 4.5, respectively. This approach has been described previously [4, 21, 22]. The mass transitions, declustering potential and collision energy of IS and test drugs with isotopologues are shown in Suppl.Table 1.
3.1.4. Sample preparation
Our ultimate choice of the Agilent deep well plate was driven by the extraction and matrix effect results as described below (3.1.5), as well the simplicity of the sample preparation without need for vacuum or dry down equipment, and lastly it was cheaper than many alternatives. The dilution of the supernatant with water resulted in an injectable sample composed of 15% acetonitrile, which was sufficiently close to the 5% acetonitrile starting mobile phase to allow injection of 5 μL while retaining appropriate peak shape of the IS.
3.1.5. Extraction recovery and matrix effect
IS recoveries and matrix effects from plasma were determined as processed with the 6 different types of 96 well extraction plates and as processed in microcentrifuge tubes, see Suppl.Table 3 and Suppl.Table 4. The Agilent deep well plate displayed relatively consistent recoveries across IS from 79–117% with reasonable precision of 20%CV or less. The Supelco Hybrid SPE had much better precision (11%CV or less), however, recoveries were lower also. Different IS experienced widely varying, and often positive, degrees of matrix effects when processed with the Agilent deep well plate. Especially the SPE plates resulted in more consistentpredominantly negative matrix effects, however with rather poor precision (up to 103%CV). Ultimately, the Agilent deep well plate was considered preferable because of the good precision, while the varying matrix effects between the IS compounds will increase the likelihood that one of these IS will appropriately function for the unknown analyte that needs to be analysed with this generic approach.
3.1.6. Selectivity and specificity
To investigate whether endogenous matrix constituents interfered with the assay, six individual batches of control, drug-free human plasma were processed and analysed according to the described procedure (section 2.4.3). Responses of IS at the working solution concentrations (section 2.4.1) were compared with the response of the blank samples. All IS were found to have a signal to noise of greater than 20% compared to control plasma samples. However, during our analysis one batch of plasma had a peak interfering with acetaminophen. We performed a product ion scan of the interfering peak and it was determined that the interfering peak was indeed acetaminophen, and not an endogenous substance by chance interfering at the acetaminophen MRM transition used. Based on this information plasma batches are now pre-screened for endogenous compounds that may interfere with detection of the IS.
3.1.7. Stability
We determined both the benchtop and freeze thaw stability of the IS working solution. The bench top stability ranged from 95% to 114% and the freeze thaw ranged from 107 to 115% (Suppl.Table 5).
3.1.8. IS Signal Stability
Three of the 8 IS had decreasing signal intensity over time (−1.5 to −14.4%) while for the remaining 5 IS, signal intensity increased (3.5 to 16.2%) (Suppl.Table 6). While on average their signal remained stable, the paclitaxel and bicalutamide had the highest signal variability, which agrees with the observation of the somewhat higher variability in matrix effect of these IS (Suppl.Table 4). The various IS options presented a moderate range of signal profiles over time, which are therefore more likely to result in an appropriate IS candidate for unknown drug analytes to be quantitated.
3.2. Performance evaluation
Analytical performance for each test analyte is reported below in a standardized format. Matrix effects are tabulated in Suppl.Table 7.
3.2.1. LMP744
Our validated method for LMP744 was linear from 10–3,000 ng/mL. Using our newly developed generic assay it was possible to quantitate LMP744 (retention time 3.0 min) from 1 to 10,000 ng/mL with erlotinib as IS (Fig. 2 and Table 1). Parent LMP744 appeared linear between 1 and 1,000 ng/mL with signs of signal saturation above 1,000 ng/mL and generic QC levels at 2, 20, 125, and 800 ng/mL showed acceptable performance. First and second isotopologue LMP744 appeared linear between 10 and 10,000 ng/mL and generic QC levels at 20, 125, 800 and 8,000 ng/mL showed acceptable performance. Established QCL and QCM were appropriately assayed with the generic assay monitoring either parent, first, or second isotopologue LMP744 mass transitions (bias −13 to +15%), while only the first and second isotopologue were able to accurately determine the QCH (bias −13 and −12%, respectively, relative to −34% for the parent transition). This is in line with the parent transition displaying less than linear response coverage at the concentration of the QCH of 2,500 ng/mL. Precision was comparable in the validated assay (4.5–5.0% coefficient of variation (CV)) and the generic assay (5.6–9.2%CV).
Fig. 2.
LMP744 residuals of the generic approach calibration (○) and QC (●) samples (A,B,C) using the MRM transition of the regular mass (A), the first isotopologue (B), or the second isotopologue (C). Associated precision (○) and bias (−) of the generic assay approach as evaluated with established assay QC samples (N=6) analysed with the generic assay, relative to the established assay (D,E,F).
Table 1.
Assay evaluation performance data for the quantitation of QCL, QCM and QCH of compounds tested with generic assay vs validated method.
| Conc. (ng/mL) | Generic result in ng/mL (Accuracy %) [%CV] | Validated result in ng/mL (Accuracy %) [%CV] | Bias (%) |
|---|---|---|---|
| 25 (QCL) | 26.1 (105) [5.7] | 23.9 (95.5) [4.5] | +9 |
| 200 (QCM) | 207 (104) [5.6] | 208 (104) [5.0] | 0 |
| 2500 (QCH) | 1843 (73.7) [5.7] | 2777 (111) [4.5] | −34 |
| 25 (QCL) | 27.3 (109) [9.2] | 23.9 (95.5) [4.5] | +14 |
| 200 (QCM) | 196 (98.0) [6.4] | 208 (104) [5.0] | −6 |
| 2500 (QCH) | 2418 (96.7) [6.3] | 2777 (111) [4.5] | −13 |
| 25 (QCL) | 27.5 (110) [9.2] | 23.9 (95.5) [4.5] | +15 |
| 200 (QCM) | 199 (99.3) [6.4] | 208 (104) [5.0] | −4 |
| 2500 (QCH) | 2453 (98.1) [6.3] | 2777 (111) [4.5] | −12 |
| 30 (QCL) | 33.5 (112) [6.0] | 29.6 (98.7) [8.0] | +13 |
| 400 (QCM) | 455 (114) [7.3] | 438 (109) [4.2] | +4 |
| 4000 (QCH) | 4032 (101) [5.2] | 4590 (115) [3.0] | −12 |
| 30 (QCL) | 29.8 (99.3) [16] | 29.6 (98.7) [8.0] | +1 |
| 400 (QCM) | 442 (111) [5.0] | 438 (109) [4.2] | +1 |
| 4000 (QCH) | 4763 (119) [7.3] | 4590 (115) [3.0] | +4 |
| 75 (QCL) | 83.8 (112) [8.6] | 78.7 (105) [2.4] | +6 |
| 750 (QCM) | 761 (101) [4.9] | 761 (101) [3.6] | 0 |
| 4000 (QCH) | 3193 (79.8) [8.0] | 4278 (107) [1.7] | −25 |
| 75 (QCL) | 84.6 (113) [7.8] | 78.7 (105) [2.4] | +8 |
| 750 (QCM) | 794 (106) [3.7] | 761 (101) [3.6] | +4 |
| 4000 (QCH) | 3975 (99.4) [8.5] | 4278 (107) [1.7] | −7 |
| 75 (QCL) | 80.4 (107) [14] | 78.7 (105) [2.4] | +2 |
| 750 (QCM) | 805 (107) [4.9] | 761 (101) [3.6] | +6 |
| 4000 (QCH) | 4485 (112) [8.7] | 4278 (107) [1.7] | +5 |
| 5 (QCL) | - | 4.97 (99.5) [5.4] | - |
| 100 (QCM) | 99.2 (99.2) [11] | 99.9 (99.9) [3.2] | −1 |
| 2500 (QCH) | 2632 (105) [6.1] | 2282 (91.3) [0.7] | +15 |
| 20 (QCL) | 20.4 (102) [7.0] | 21.7 (109) [0.5] | −6 |
| 200 (QCM) | 230 (115) [4.0] | 200 (99.9) [2.3] | +15 |
| 800 (QCH) | 900 (112) [2.9] | 773 (96.6) [4.6] | +16 |
| 5 (QCL) | 4.9 (98.5) [14.4] | 5.3 (106) [15] | −7 |
| 150 (QCM) | 148 (98.6) [5.0] | 164 (109) [4.3] | −10 |
| 4000 (QCH) | 3703 (92.6) [15] | 3975 (99.4) [3.8] | −7 |
| 6 (QCL) | 9.0 (150) [28.9] | 6.5 (109) [2.0] | +38 |
| 20 (QCM) | 22.3 (112) [20.4] | 21.4 (107) [2.6] | +4 |
| 600 (QCH) | 560 (93.3) [8.2] | 584.2 (97.4) [4.6] | −4 |
| 6 (QCL) | 5.9 (98.1) [12.1] | 6.5 (109) [2.0] | −10 |
| 20 (QCM) | 19.4 (96.8) [15.5] | 21.4 (107) [2.6] | −9 |
| 600 (QCH) | 574 (95.7) [10.9] | 584.2 (97.4) [4.6] | −2 |
Six QCs were analyzed at each level using both the generic assay and the validated assays. The percent difference of the means between the generic assay and the validated assays at each concentration was calculated.
3.2.2. Olaparib
Our validated method for olaparib was linear from 10–5,000 ng/mL. Using our newly developed generic assay it was possible to able to quantitate olaparib (retention time 3.13 min) from 3 to 10,000 ng/mL with abiraterone as IS, see Fig. 3 and Table 1. Parent olaparib appeared linear between 3 and 3,000 ng/mL with signs of signal saturation at 10,000 ng/mL and generic QC levels at 2, 20, 125, 800 and 8,000 ng/mL showed acceptable performance. First isotopologue olaparib appeared linear between 10 and 10,000 ng/mL and generic QC levels at 20, 125, 800 and 8,000 ng/mL showed acceptable performance. While established QCs were appropriately assayed with the generic assay monitoring parent olaparib mass transition (bias −12 to +13%), the first isotopologue was more accurate (bias +1 to +4%). This is in line with the first isotopologue providing linear response coverage across the QC concentration range. Precision was slightly better in the validated assay (3.0–8.0%CV) relative to the generic assay (5.0–16%CV).
Fig. 3.
Olaparib residuals of the generic approach calibration (○) and QC (●) samples (A,B,C) using the MRM transition of the regular mass (A), or the first isotopologue (B). Associated precision (○) and bias (−) of the generic assay approach as evaluated with established assay QC samples (N=6) analysed with the generic assay, relative to the established assay (C,D).
3.2.3. Cabozantinib
Our FDA validated method for cabozantinib was linear from 50–5,000 ng/mL. Using our newly developed generic assay it was possible to quantitate cabozantinib (retention time 3.57 min) from 1 to 10,000 ng/mL by using both the [M+H]+ and the isotopologue MRM channels transition, see Suppl.Figure 1 and Table 1. We used erlotinib as the IS for cabozantinib as they coeluted. Parent cabozantinib appeared linear between 1 and 1,000 ng/mL with signs of signal saturation at higher concentrations and generic QC levels at 2, 20, 125, and 800 ng/mL showed acceptable performance. First isotopologue cabozantinib appeared linear between 3 and 3,000 ng/mL and generic QC levels at 20, 125, and 800 ng/mL showed acceptable performance. Second isotopologue cabozantinib appeared linear between 30 and 10,000 ng/mL and generic QC levels at 125, 800, and 8,000 ng/mL showed acceptable performance. Established QCL and QCM were appropriately assayed with the generic assay monitoring either parent, first, or second isotopologue cabozantinib mass transitions (bias 0 to +8%), while only the first and second isotopologue were able to accurately determine the QCH (bias −7 and +5%, respectively, relative to −25% for the parent transition). This is in line with the parent transition displaying less than linear response coverage at the concentration of the QCH of 4,000 ng/mL. Precision was better in the validated assay (1.7–3.6%CV) relative to the generic assay (3.7–14%CV).
3.2.4. Triapine
Our validated method for triapine was linear from 3–3,000 ng/mL. Our newly developed generic assay was able to quantitate triapine (retention time 1.4 min), but performance characteristics were not optimal, see Suppl.Figure 2 and Table 1. Though triapine appeared linear between 10 and 10,000 ng/mL using acetaminophen as IS, several calibrators had a bias of more than 20%, and generic QCs also showed bias (−1.9% to −38.1%) and variability (11.4–20.3%CV). Even so, stablished QCs at 100 and 2,500 ng/mL were appropriately assayed with the generic assay (bias −1 to +15%). Precision was better in the validated assay (0.7–5.4%CV) relative to the generic assay (6.1–11%CV). The variable performance of the generic assay applied to triapine may be due to the polar nature of triapine, which eluted at 0.6 min which is near the void time (0.5 min).
3.2.5. Ixabepilone
Our validated method for ixabepilone was linear from 1–1,000 ng/mL. Using our newly developed generic assay it was possible to quantitate ixabepilone (retention time 3.55 min) from 10 to 10,000 ng/mL with abiraterone as IS, see Suppl.Figure 3 and Table 1. Ixabepilone appeared linear between 10 and 10,000 ng/mL with loss of linearity between 3 and 10 ng/mL, and generic QC levels at 20, 125, 800 and 8,000 ng/mL showed acceptable performance while 2 ng/mL was undetectable. With the exception of 2 ng/mL (undetectable), established QCs at 20, 200, and 800 ng/mL were appropriately assayed with the generic assay (bias −6.2 to +16%). Precision was slightly better in the validated assay (0.5–4.6%CV) relative to the generic assay (2.9–7.0%CV).
3.2.6. Berzosertib
Our validated method for berzosertib was linear from 3–5,000 ng/mL. Using our newly developed generic assay it was possible to quantitate berzosertib (retention time 3.09 min) from 10 to 10,000 ng/mL with abiraterone as IS, see Suppl.Figure 4 and Table 1. Berzosertib appeared linear between 10 and 10,000 ng/mL with an endogenous peak that prevented quantitation at 1 and 3 ng/mL. Generic QC levels at 20, 125, 800 and 8,000 ng/mL showed acceptable performance. Established QCs at 5, 150, and 4000 ng/mL were appropriately assayed with the generic assay (bias −6.6 to −9.9%). Precision was similar in the validated (3.8–15%CV) and the generic assay (5–15%CV).
3.2.7. Eribulin
Our validated method for eribulin was linear from 0.1–10 and 10–1,000 ng/mL. Using our newly developed generic assay it was possible to quantitate eribulin (retention time 3.52 min) from 10 to 3,000 ng/mL (upper concentration limited by available stock concentration solution) with erlotinib as IS. However, performance characteristics were not optimal, see Suppl.Figure 5A and C and Table 1. The lower limit of quantitation was 10 ng/mL at best, and established QCM precision was more than 20%. We decided to reanalyse the samples with an increase of injection volume of 20 μL instead of the standard 5 μL. While peak shape was not compromised, this not only reduced the lower limit of quantitation to 3 ng/mL, but also improved the assay performance, see Suppl.Figure 5B and D and Table 1. Generic QC levels at 20, 125, and 800 ng/mL showed acceptable performance. Established QCs at 6, 20, and 600 ng/mL (0.2 ng/mL was not studied as it was below the generic range) were appropriately assayed with the generic assay (bias −10 to −2%). Precision was better in the validated (2.0–4.6%CV) than in the generic assay (11–16%CV).
3.3. Application of the assay
The assay range was adjusted based on expected rogaratinib concentrations. Rogaratinib could be quantitated with the generic assay approach (retention time 3.2 min). The assay had acceptable performance based on QCs at 150, 1,500, and 15,000 ng/mL (N=6) with 107.1–109.7% accuracy and <10.6%CV precision. Data are shown in Fig. 4 with plasma and tumor concentrations in the same order of magnitude. Doses of 27, 55, and 164 mg/kg resulted in plasma Cmax 710, 2560, and 8348 ng/mL; half-life 3.8, 4.8, and 4.6 h; and apparent clearance 6.4, 3.1, and 2.4 L/h/kg, respectively, suggesting a more than proportional increase in exposure with dose. Samples at 24 h did not have detectable concentrations other than the highest dose level (extrapolated calculations in the single digit ng/mL concentrations), confirming non-linear PK, and suggesting that for definitive pharmacokinetics, more densely sampled studies will be needed.
Fig. 4.
Rogaratinib plasma (A) and tumor (B) concentrations (mean±SD) after oral dosing of mice at 27 mg/kg (□), 55 mg/kg (Δ), or 164 mg/kg (○).
4. Conclusion
To our knowledge, this is the first generic approach method that is described in great detail, includes multiple IS candidates, and is extensively evaluated with test agents against established and validated drug-specific assay methodology.
The final assay is comprised of a simple sample preparation. While each plate method could have been further optimized for a specific analyte to achieve lower matrix effects and better performance for that specific analyte, that was not the objective for our generic assay. Our generic assay could quantitate drugs down to at minimum 10 ng/mL, and often down to 1 ng/mL utilizing a 50 μL sample volume. Linearity was assured over 3 orders of magnitude, but no more, and isotopologues were successfully utilized to shift the linear portion of the assay across the concentration range without having to reanalyze samples. Multiple IS allowed for empirical choosing of the best IS for a given compound, though in our experience, erlotinib or abiraterone functioned appropriately for most test agents, while usually multiple IS would have performed similarly, providing redundancy. In validating our generic assay approach with 7 test agents, established assay QCs could be quantitated with precisions within 15% CV, and bias of up to 16%, as long as the appropriate isotopologue was chosen.
Previously, different levels of assays have been described for screening-type pharmacokinetic needs [14], each with specific performance requirements. In addition, the recent FDA guidance for bioanalytical method validation also details criteria by which to judge whether assay results may be utilized [23]. The minimal assay performance required to declare a batch passable may be set depending on the objective and needs. It is worth noting that this generic approach may be utilized as a starting point for agent-specific assays.
In conclusion, the developed generic assay approach promises to be a useful bioanalytical tool, and has already been applied with acceptable performance to pharmacokinetic plasma and tissue homogenate samples with rogaratinib, dabrafenib, afatinib, and several agents in early preclinical development (data not shown).
Supplementary Material
Highlights.
There is a lack of well-characterized generic assay approaches
A generic assay approach for small molecule bioanalysis is feasible
Linearity over 3 orders of magnitude may be extended by use of isotopologues
Eight internal standard candidates are available for use
Seven test agents with established assays were used to validate the generic approach
Acknowledgements
Funding
Support: Grants R50CA211241, U24CA247643, UM1CA186690, Contract HHSN261201600022I, control number N02CM-2016-00022, and contract HHSN261201500003I, (NCI). This project used the UPMC Hillman Cancer Center’s Cancer Pharmacokinetics and Pharmacodynamics Facility (CPPF) and the City of Hope Comprehensive Cancer Center’s Analytical Pharmacology Core Facility and was supported in part by awards P30-CA47904 and P30-CA033572. Declarations of interest: none.
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. NCI- Frederick is accredited by AAALACi and follows the Public Health Service Policy on the Care and Use of Laboratory Animals. All animals used in this research project were cared for and used humanely according to the following policies: The U.S. Public Health Service Policy on Humane Care and Use of Animals (1996); the Guide for the Care and Use of Laboratory Animals Eighth Edition; and the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (1985).
Footnotes
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