Abstract
We have developed and validated a novel LC–MS/MS method for the simultaneous quantification of LB-100 and its active metabolite, endothall, in human plasma following solid-phase extraction. LB-105 and endothall-D6 were used as internal standards. Chromatographic separation was achieved on a Hypercarb™ column using 5 mM (NH4)2CO3 and 30:70 (v/v) 100 mM (NH4)2CO3:acetonitrile as mobile phases. Detection was performed via positive electrospray ionization mode with multiple reaction monitoring. The assay exhibited linearity in the concentration range of 2.5–500 ng/ml for both analytes. Intra- and inter-assay precision and accuracy were within ±11%. LB-100 and endothall recoveries were 78.7 and 86.7%, respectively. The validated LC–MS/MS method enabled the accurate measurement of LB-100 and endothall in patient samples from an ongoing clinical trial (NCT04560972).
Keywords: chromatography, endothall, human plasma, LB-100, LC–MS, mass spectrometry, oncology, pharmacokinetics, validation
Protein phosphatase 2A (PP2A) in an essential enzyme involved in regulating various cellular processes, including cell survival, apoptosis, mitosis and the cellular response to DNA damage [1]. It has long been accepted as a potential target for anticancer drugs. Previous studies have shown that PP2A inhibition exhibits potent chemo- and radio-sensitizing properties; however, because of the multifunctionality of the PP2 family, pharmacological inhibitors of these enzymes are considered too toxic to be used as anticancer treatments [2]. To overcome the toxicities of naturally produced compounds, a potent small-molecule inhibitor of PP2A called LB-100 (3-(4methylpiperazine-carbonyl)-7-oxalobicyclo[2.2.1]heptane-2-carboxylic acid; NSC D753810) was developed.
Preclinical studies have demonstrated the potential of LB-100 as a novel anticancer drug in the treatment of human malignancies including glioblastoma [3–5], sarcoma [6], chordoma [7], breast cancer [8], meningioma [9], leukemia [10], pancreatic cancer [11], epidermoid carcinoma [12] and lung cancer [13]. Mounting data have demonstrated that LB-100 has single-agent activity and potentiates the activity of standard cytotoxic agents and/or radiation without enhancing toxicity [1,14]. The mechanism of potentiation appears to increase drug penetration, overcome cancer cell senescence, induce cellular differentiation in progenitor cells and promote mitotic catastrophe and apoptosis [3,6]. Moreover, previous studies have shown that LB-100 is an active anticancer agent both in itself and by its in vivo conversion to endothall, which increases the effective duration of inhibition of PP2A [5,15,16].
Due to its exceptional mechanism of action and its capacity to enhance the efficacy of a wide range of anticancer agents, LB-100 has prompted the initiation of multiple clinical investigations. Up to April 2023, a total of four clinical trials have been conducted in the USA (www.clinicaltrials.gov), demonstrating the broad scope of its application in both hematological and solid malignancies. Thus there is a clinical need for therapeutic monitoring of LB-100 along with its metabolite, endothall. In particular, there is a clinical trial investigating the side effects and best dose of LB-100 when given together with standard chemotherapy drugs for the treatment of untreated extensive-stage small-cell lung cancer being conducted by City of Hope Medical Center [17]. To precisely assess the pharmacokinetics of LB-100 and its metabolite, it is necessary to develop a robust analytical method that offers selectivity, simplicity, sensitivity, minimal sample volume and expedited analysis time.
Developing a single method to analyze LB-100 and endothall presents several challenges due to their ex vivo stability and different polarity, leading to differential chromatographic behavior and ionization effects. Previous strategies for determining LB-100 and endothall in biological matrices involved separate extraction and LC–MS/MS assays. For example, LC–MS/MS assays for LB-100 and endothall in rat plasma and tissue were reported separately in the Lixte Biotechnology patent [16], with LLOQ at 3 and 6 ng/ml for LB-100, and 20 ng/ml for endothall in rat plasma, brain and liver homogenate. Similarly, Quang et al. [18] reported two separate LC–MS/MS assays for LB-100 and endothall in human plasma to support a previous phase I clinical trial [14]. While various analytical methods are available for analyzing endothall in drinking water and soil [19–21], applying those methods to biological matrices is difficult. Guo et al. reported an LC–MS/MS method for quantitating cantharidic acid in rat blood using endothall as the internal standard (IS) [22].
To our knowledge, this is the first report of a method that allows the simultaneous determination of LB-100 and endothall using LC–MS/MS in human plasma. Our LC–MS/MS approach offers exceptional sensitivity (2.5 ng/ml), demands a small sample volume (100 μl) and provides a short turnaround time. We successfully validated this bioanalytical method and applied it to the quantitative analysis of LB-100 and endothall in patient plasma samples received from a subject enrolled in an ongoing clinical trial.
Materials & methods
Chemicals & reagents
The reference standards of LB-100 and LB-105 were provided by Lixte Biotechnology Holdings, Inc. (CA, USA). Endothall and endothall-D6 were obtained from Alfa Aesar (MA, USA) and C/D/N Isotopes Inc. (Pointe-Claire, Canada), respectively. Deionized water was generated in-house using a Milli-Q water purification system obtained from Millipore (MA, USA). HPLC-grade acetonitrile (ACN), HPLC-grade methanol (MeOH), DMSO, sodium hydroxide (NaOH) and ammonium carbonate (NH4)2CO3 were purchased from Thermo Fisher Scientific (PA, USA). Six lots of pooled sodium heparinized human blank plasma were purchased from Innovative Research (MI, USA) and were stored at -20°C prior to use.
Calibration curves
The individual stock solutions of LB-100 (1.00 mg/ml) and LB-105 (1.00 mg/ml) were prepared by dissolving the chemicals in 10 mM NaOH and were kept at -20°C before use. The stock solutions of endothall (1.00 mg/ml) and endothall-D6 (1.00 mg/ml) were prepared individually in DMSO and kept at -20°C before use. At least two batches of LB-100 and endothall stock solution should be prepared, one used for calibrators and another used for quality controls (QCs). The dilution solution for preparing working standard solutions contained MeOH:100 mM (NH4)2CO3 (50:50, v/v). The diluted working standard solutions were used for the preparation of the calibration curve and QC samples.
Before spiking, 50 mM NaOH-treated blank human plasma was screened to ensure the absence of endogenous interference at the retention times of the analytes. An eight-point standard curve of LB-100 and endothall was prepared by mixing the blank plasma with an appropriate amount of LB-100 and endothall working standard solutions with a spiked ratio at 1:10. The calibration curve ranged from 2.5 to 500 ng/ml for both endothall and LB-100. Similarly, QC samples were prepared at three concentration levels of 7.5, 40 and 400 ng/ml.
Sample preparation
Patient plasma samples treated with 50 mM NaOH were prepared by spiking 10 μl of the dilution solution into 100 μl of each patient’s plasma. This step was performed to match the sample matrix with those of the plasma calibrators and QCs.
Single blank plasma was prepared by spiking 10 μl of the dilution solution into 100 μl of pooled blank human plasma. Similarly, double blank plasma was prepared by spiking 15 μl of the dilution solution into 100 μl of pooled blank human plasma.
Prior to mixing plasma samples with 1 ml of 10 mM NaOH, 5 μl of IS working solution (5 μg/ml endothall-D6 and 0.5 μg/ml LB-105) was added to each of the above plasma samples (single blank, calibrators, QCs and patient samples) except the double blank plasma, and mixed well.
After a 2-min vortex, the sample mixture was loaded into an Oasis MAX 1-cc Vac Cartridge (30 mg, 30 μm; Waters, MA, USA) that was preconditioned with: 0.5 ml of MeOH, followed by 0.5 ml of 10 mM NaOH and 0.5 ml of water. The extraction cartridge was washed with 1 ml of water followed by 1 ml of 30% MeOH and then MeOH. Analytes were eluted with 0.4 ml of 1% w/v (NH4)2CO3 /MeOH and dried in a Thermo Reacti III system (Thermo Fisher Scientific, MA, USA) at 25°C under nitrogen gas. The resulting residue was reconstituted in 100 μl of the dilution solution, and 5 μl of the eluent was injected into the LC–MS/MS system.
Instrumentation
The LC–MS/MS system consisted of a Prominence HPLC system (Shimadzu, Kyoto, Japan) interfaced to an AB SCIEX QTRAP® 5500 system (Applied Biosystems Sciex, Concord, Canada) using Analyst v. 1.6 software. The system was operated in the ESI positive multiple reaction monitoring (MRM) mode. It was tuned by 100 ng/ml solution of each analyte or IS in 50% ACN for both the compound- and the source-dependent parameters. The source/gas parameters were set as follows: curtain gas at 30, collision assisted dissociation gas at medium; ionization voltage at 4500 V; source temperature at 700; sheath gas at 60; desolvation gas at 60. The compound parameters including declustering potential, entrance potential, collision energy and cell exit potential were fine-tuned by adjusting the voltage range. The signal intensity of the compound of interest was monitored by using the automated ‘edit ramp’ function. Detailed information regarding the optimized mass transitions and MRM parameters can be found in Table 1.
Table 1. . Mass spectrometry parameters.
| Analyte | Q1 | Q3 | DP (V) | CE (V) | EP (V) | CXP (V) |
|---|---|---|---|---|---|---|
| LB-100 | 269.2 | 123.1 | 105 | 55 | 5 | 15 |
| LB-105 (LB-100 IS) | 283.2 | 123.0 | 105 | 50 | 5 | 15 |
| Endothall | 169.1 | 123.0 | 105 | 12 | 5 | 15 |
| Endothall-D6 (endothall IS) | 175.1 | 147.2 | 105 | 9 | 5 | 15 |
Q1 = Quadrupole mass filter 1 (Q1) and 3 (Q3).
CE: Collision energy; CXP: Collision cell exit potential; DP: Declustering potential; EP: Entrance potential; IS: Internal standard.
The separation was achieved using a Hypercarb 3.0 μ 100 × 2.1 mm column preceded by a Hypercarb guard column (both Thermo Fisher Scientific). The column temperature was maintained at 75°C and the flow rate was 0.5 ml/min. Mobile phase A was 5 mM (NH4)2CO3 and mobile phase B was 30:70 (v/v) 100 mM (NH4)2CO3:ACN. The following gradient program was used: 2% to 20% B (0.0–1.0 min), 20% to 95% B (1.0–3.0 min), 95% B (hold 3.0–4.0 min), 95% to 2% B (4.0–4.1 min), 2% B (hold 4.1–6.0 min). The total run time was 6 min. The autoinjector temperature was maintained at 5°C and the injection volume was 5 μl.
Validation
The LC–MS/MS method developed was validated in human plasma in terms of linearity, selectivity, LLOQ, accuracy and precision, recovery, matrix effect and stability for both short-term sample processing and long-term sample storage. Acceptance criteria were ±20% for LLOQ and ±15% for all other levels following the US FDA (2019) guidance for industry on bioanalytical method validation [23].
Calibration curves in human plasma were established by plotting the peak area ratio of each analyte to its respective IS against standard concentrations. Eight nonzero plasma calibrators in duplicates were prepared over three different batches using a linear 1/concentration squared weighted regression algorithm (1/x2). The LLOQ was defined as the lowest concentration of LB-100 and endothall plasma calibrator on the calibration curve with accuracy and precision within ±20%. Accuracy was determined by percentage relative error, while precision was assessed using %CV.
The selectivity of this assay was evaluated by comparing LLOQ level and blank from different sources (six individual blank plasmas). Any interferents from blank matrix at the same retention times and mass transitions of the analytes should be less than 20% response of LLOQ.
For the assessment of inter-assay precision and accuracy, five parallel analyses of five identical QC samples were conducted at each of four QC concentrations (2.5, 7.5, 40 and 400 ng/ml). The intra-assay precision and accuracy were determined through five replicate analyses of each QC sample. Additionally, a dilution study was conducted, where the dilution QCs (200 ng/ml) were prepared by tenfold dilution with the pooled human blank plasma.
The extraction recovery of the target analytes from plasma at low, mid and high levels (7.5, 40 and 400 ng/ml) was evaluated by comparing the extracted analyte responses (spiked before extraction) with analyte responses spiked into extracted blank samples (spiked after extraction), which served as the 100% recovery for reference. The matrix effect was assessed by comparing the responses of the analytes spiked into extracted blank samples (spiked after extraction) with the responses of the analytes in the dilution solution (MeOH:100 mM (NH4)2CO3, 50:50 v/v).
Stability studies were conducted for analyte stock and working solutions (1.00 mg/ml and 1.00 μg/ml) and plasma low- and high-concentration QC samples (7.5 and 400 ng/ml). Short-term stability was assessed by storing samples on the benchtop at 23°C and in the autosampler at 4°C for 24 h. Freeze–thaw stability was evaluated during three cycles of freezing at -20°C for at least 24 h and thawed at room temperature (23°C). Long-term stability was conducted by storing samples at -20°C for 30 days. The stabilities of analytes were calculated by comparing the mean peak area ratio of analyte to the corresponding IS in the test sample with those of freshly prepared samples, considered as percentages. All experiments were conducted in five replicates.
Method application
The LC–MS/MS method developed was applied for the measurement of LB-100/endothall in patient plasma samples collected from a phase Ib open-label study of LB-100 in combination with carboplatin/etoposide/atezolizumab in untreated extensive-stage small-cell lung carcinoma [18], where patients were given LB-100 by a 15-min intravenous infusion on days 1 and 3. Blood samples were collected in heparinized tubes at the following time points: pre-dose, 0, 0.25, 0.5, 1, 2, 3, 4 and 8 h after the end of intravenous infusion; 5 ml of venous blood was drawn into a chilled sodium heparin collection tube and kept on ice until the plasma was separated and aliquoted (two aliquots) into appropriately labeled polypropylene tubes containing 0.5 N NaOH. For every 1.0 ml of plasma aliquoted, 0.1 ml of 0.5 N NaOH was added. Plasma samples were stored at -70°C before analysis.
During the analysis, patient samples were processed alongside a series of calibrators including double blank, single blank and eight nonzero calibrators, and a set of QCs at low, medium and high concentrations (7.5, 40 and 400 ng/ml). The samples and standards were prepared and processed following the procedures described in the ‘sample preparation’ section, and subsequently analyzed by the validated method.
Results & discussion
In this study we aimed to develop and validate a robust assay that is straightforward, fast and highly sensitive for the simultaneous extraction and quantification of LB-100 and endothall. This assay was specifically designed to be suitable for determining the pharmacokinetics of these compounds in clinical studies. During the method development, different approaches were explored to optimize detection parameters, chromatography and sample extraction.
Mass spectrometric detection
For the identification and quantification of LB-100 and the IS LB-105, positive ESI was selected because of their higher tendency to generate protonated ions rather than deprotonated ions. The predominate molecular ions of LB-100 and LB-105 were observed at m/z 269.2 and 283.2, respectively. These protonated molecular ions could be further fragmented into product ions through collision with nitrogen gas, as shown in Figures 1 & 2 (A & B). The product ion at m/z 123.1 was finally selected for the quantitation of LB-100 due to its minimal background noise and high signal-to-noise ratio, surpassing other product ions. Similarly, m/z 283.2 > 123.1 was chosen for the IS LB-105.
Figure 1. . Chemical structures with fragmentation.
(A) LB-100, (B) the internal standard LB-105, (C) endothall and (D) the internal standard endothall-D6.
Figure 2. . Mass spectra.
(A) LB-100, (B) LB-105, (C) endothall and (D) endothall-D6.
For endothall and endothall-D6, both negative and positive ionization modes were tested. The results indicated a stronger signal response in the positive ionization mode than the negative mode. Consequently, the precursor ions [M–H2O+H]+ at m/z 169.1 for endothall and m/z 175.1 for the isotope IS endothall-D6 were used. Chemical structures with fragmentation patterns and product ions of endothall and endothall-D6 are shown in Figures 1 & 2 (C & D). The predominant product ion at m/z 123.1 was selected for endothall, and m/z 147.1 was selected for endothall-D6. To optimize signal responses, the ‘auto-tune’ function of AB Sciex Analyst software (v. 1.6.1) was utilized. The optimized MRM parameters were described in the ‘instrumentation’ section above.
Liquid chromatographic separation
First, addition of NaOH was recommended in preparation of the stock solution and plasma samples for increasing the solubility and stability of LB-100. In this study, degradation of LB-100 was observed when we prepared a solution of LB-100 with unbuffered water. For better understanding of factors that affect the stability of LB-100, the stability of LB-100 in different pH conditions was investigated by incubating LB-100 with either 1% formic acid, 10 mM NH4CH3CO2 (pH 5.0), 10 mM (NH4)2CO3 (pH 9.2) or 10 mM NaOH at room temperature. Degradation of LB-100 was observed under acidic conditions (10 mM NH4CH3CO2 or 1% formic acid), but there was no loss of LB-100 under basic conditions (10 mM (NH4)2CO3 or 10 mM NaOH), which indicated that maintaining a basic pH was key to preventing hydrolysis during the entire process of LB-100 detection. Therefore, NaOH was proposed to be added to the LB-100 stock solution and plasma samples. For the same purpose, (NH4)2CO3 (pH 9.2) was suggested to be used in the dilution solution and mobile phase.
The goal of this work was to develop and validate a sensitive and simple method for the simultaneous quantification of LB-100 and its metabolite endothall. After basic conditions were chosen for preventing degradation of LB-100 in this assay, another challenge we encountered stemmed from the extremely hydrophilic characteristics of endothall at pH > 6. In this work, several reverse-phase columns including a Waters Acquity UPLC® BEH C18 (2.1 × 150 mm, 1.7 μm), a Waters X-Bridge AQ (2.0 × 100 mm, 3.5 μm) and a Phenomenex Kinetex C18 (4.6 × 100 mm, 2.6 μm) were tested; however, none of them was able to retain endothall even with 100% aqueous phase. A mixed-mode chromatography column Imtakt SS-C18 (100 × 3 mm) was also tested due to its unique combination of both reversed-phase and ion-exchange mode; however, although a reasonable retention time of endothall was achieved on this column, it failed to provide good detection sensitivity for LB-100. Eventually, a Thermo Hypercarb (2.1 × 100 mm, 3.0 μm) was chosen because it gave a reasonable retention time and good separation of LB-100 and endothall as well as their ISs.
Further modification in chromatography conditions increased the signal intensity of analytes and robustness of retention times. Unlike conventional reversed-phase columns, Hypercarb is a porous graphitic carbon column containing weak anion exchange sites on its surface, which provides unique retention and separation of polar compounds. The unique retention mechanism of Hypercarb requires a different method development strategy. In this study, different mobile-phase combinations were tried out, and ammonium carbonate at pH 9.2 in combination with ACN provided sharper peak shape and better resolution. Moreover, we found that ionic strength strongly affected the retention and separation of analytes. A gradient with increasing concentrations of (NH4)2CO3 and ACN helped in improving signal response and stabilizing the retention times of analytes. The optimal separation was achieved using mobile phases consisting of 5 mM (NH4)2CO3 (mobile phase A) and 30:70 (v/v) 100 mM (NH4)2CO3:ACN (mobile phase B), with the following gradient program: 2% to 20% B (0–1.0 min), 20% to 95% B (1.0–3.0 min), 95% B (hold 3.0–4.0 min), 95% to 2% B (4.0–4.1 min), 2% B (hold, 4.1–6.0 min). The gradient flow rate was 0.5 ml/min, and the column oven temperature was optimized to 75°C in order to reduce column pressure and achieve symmetry of analyte peaks. Under optimized assay conditions, the retention times for endothall, endothall-D6, LB-100 and LB-105 are 1.6, 1.6, 2.9 and 3.2 min, respectively (Figure 3).
Figure 3. . Representative multiple reaction monitoring chromatograms of human plasma.
(A) Double blank. (B) 2.5 ng/ml LB-100 and endothall in plasma. (C) Plasma sample from a patient 8 h after an infusion on day 3. (D) Single blank.
Sample preparation
Sample extraction is performed to remove the endogenous interference in plasma constituents and enrich the analytes. Protein precipitation using ACN or MeOH gave strong interferences from endogenous substances in plasma, which significantly suppressed the sensitivity of endothall and precluded the determination of endothall at concentrations below 10 ng/ml.
To eliminate the interfering components, solid-phase extraction (SPE) was utilized in this assay because it offered a cleaner extract compared with protein precipitation methods. Oasis MAX cartridges (30 mg, 1 cc) were selected as they have mixed-mode anion exchange sorbents. Considering both endothall and LB-100 are acidic compounds, alkalized plasma samples with NaOH helped retain analytes on SPE absorbent. Given that LB-100 is not stable under acidic conditions, instead of acidifying the elution solvent we increased ionic strength to help elute the analytes. To further increase the sensitivity of this assay, the extract was evaporated to dryness and reconstituted in the dilution solution. We found that the final procedure of the sample pretreatment achieved satisfactory extraction recovery as well as sufficient sensitivity for both endothall and LB-100.
Selectivity & LLOQ
Figure 3A shows the absence of significant interferents at the retention times and mass transitions of all analytes from various plasma matrices, including six individual plasmas and the pooled plasma, as well as the predosed plasma from patients on day 1 in a phase Ib clinical trial.
The method achieved LLOQ at 2.50 ng/ml for both LB-100 and endothall in human plasma (Figure 3B). The precision and accuracy at LLOQ were assessed using five replicates with one injection per sample in each lot. The results, summarized in Table 2, revealed the accuracy and the precision to be within ±13% for LB-100 and within ±14% for endothall, which are lower than the industry limits recommended by the FDA (within ±20%) [23].
Table 2. . Accuracy and precision of LB-100 and endothall at the lower limit in six individual lots of human plasma matrices.
| Analyte | Plasma matrix | Nominal concentration (ng/ml) | Mean measured (ng/ml) | SD (ng/ml) | Precision† (%CV) | Accuracy‡ (%RE) |
|---|---|---|---|---|---|---|
| LB-100 | Lot 1 | 2.50 | 2.36 | 0.19 | 8 | -6 |
| Lot 2 | 2.50 | 2.58 | 0.34 | 13 | 3 | |
| Lot 3 | 2.50 | 2.25 | 0.29 | 13 | -10 | |
| Lot 4 | 2.50 | 2.33 | 0.12 | 5 | -7 | |
| Lot 5 | 2.50 | 2.35 | 0.15 | 6 | -6 | |
| Lot 6 | 2.50 | 2.23 | 0.14 | 6 | -11 | |
| Endothall | Lot 1 | 2.50 | 2.53 | 0.14 | 6 | 1 |
| Lot 2 | 2.50 | 2.39 | 0.33 | 14 | -4 | |
| Lot 3 | 2.50 | 2.51 | 0.32 | 13 | 1 | |
| Lot 4 | 2.50 | 2.35 | 0.21 | 9 | -6 | |
| Lot 5 | 2.50 | 2.49 | 0.13 | 5 | -1 | |
| Lot 6 | 2.50 | 2.40 | 0.15 | 6 | -4 |
%CV = (SD/mean) × 100%
%RE = [(measured – nominal)/nominal] × 100%
CV: Coefficient of variation; RE: Relative error; SD: Standard deviation.
Matrix effect & recovery
To assess the matrix effect, we explored different sample preparation approaches, including protein precipitation. As we discussed in the ‘sample preparation’ section, protein precipitation methods resulted in strong interferences from endogenous substances present in plasma, leading to significant signal suppression and compromising the sensitivity of endothall quantification. To overcome these challenges, we developed a SPE approach which effectively minimized the signal suppression due to the matrix effect. By implementing the optimized sample preparation method, we achieved accurate quantification of endothall down to a low concentration of 2.5 ng/ml, which was not feasible with protein precipitation methods. The absence of significant interferents in the chromatogram, as depicted in Figure 3A, further supports the minimal matrix interference of our LC–MS/MS method.
Furthermore, we evaluated the matrix effect using extracted bank plasma samples spiked with LB-100 and endothall. Table 3 presents the absolute matrix effect and the IS-normalized matrix effect. Importantly, our optimized method resulted in negligible matrix interference, as evidenced by the absolute matrix effect ranging from 0.80 to 0.98 for LB-100 and from 0.92 to 1.04 for endothall. After normalizing to the IS, the IS-normalized matrix effect remained within the range 0.99–1.05 for LB-100 and 0.94–1.00 for endothall, further confirming the minimal impact of the plasma matrix on the quantitation of both analytes.
Table 3. . Matrix factor of LB-100 and endothall in pooled human plasma.
| Analyte | [Analyte] (ng/ml) | MFAnalyte ± SD | MFIS ± SD | IS-normalized MF ± SD |
|---|---|---|---|---|
| LB-100 | 7.50 | 0.81 ± 0.04 | 0.79 ± 0.02 | 1.04 ± 0.04 |
| 40.0 | 0.80 ± 0.02 | 0.77 ± 0.01 | 1.05 ± 0.01 | |
| 400 | 0.98 ± 0.05 | 0.99 ± 0.05 | 0.99 ± 0.03 | |
| Endothall | 7.50 | 1.04 ± 0.01 | 1.10 ± 0.03 | 0.94 ± 0.03 |
| 40.0 | 1.02 ± 0.04 | 1.07 ± 0.05 | 0.96 ± 0.01 | |
| 400 | 0.92 ± 0.04 | 0.92 ± 0.06 | 1.00 ± 0.03 |
MFAnalyte = (mean peak area of analyte in extracted plasma matrix)/(mean peak area of analyte in mobile phase); MFIS = (mean peak area of IS in extracted plasma)/(mean peak area of IS in mobile phase); and IS-normalized MF = MFAnalyte /MFIS.
IS: Internal standard; RE: Relative error; SD: Standard deviation.
QC samples were prepared by adding the analytes to the plasma matrix before and after extraction, allowing the evaluation of recovery efficiency. The percentage recovery was determined by comparing the average peak area ratios of the analytes to IS in the respective QC samples. As shown in Table 4, the absolute recoveries for LB-100 and endothall ranged from 76 to 81% and from 81 to 91%, respectively. The IS-normalized recoveries of LB-100 and endothall were consistent between 97 and 105%. These results indicated that our sample preparation protocol effectively recovered the analyte and the IS from human plasma.
Table 4. . Recovery of LB-100 and endothall in pooled human plasma.
| Analyte | [Analyte] (ng/ml) | RecoveryAnalyte ± SD | RecoveryIS ± SD | IS-normalized recovery ± SD |
|---|---|---|---|---|
| LB-100 | 7.50 | 81 ± 11 | 80 ± 8 | 101 ± 7 |
| 40.0 | 76 ± 10 | 72 ± 5 | 105 ± 6 | |
| 400 | 79 ± 11 | 81 ± 8 | 98 ± 6 | |
| Endothall | 7.50 | 88 ± 9 | 90 ± 3 | 98 ± 10 |
| 40.0 | 91 ± 5 | 94 ± 5 | 97 ± 2 | |
| 400 | 81 ± 3 | 79 ± 8 | 104 ± 11 |
RecoveryAnalyte = [(mean peak area of analyte in plasma matrix)/(mean peak area of analyte in extracted plasma matrix)] × 100%; RecoveryIS = [(mean peak area of IS in plasma matrix)/(mean peak area of IS in extracted plasma matrix)] × 100%; and IS-normalized recovery = (RecoveryAnalyte/ RecoveryIS) × 100%.
IS: Internal standard; RE: Relative error; SD: Standard deviation.
Linearity
A linear regression analysis was performed using 1/x2 as a weighting factor to plot the peak area ratio of analyte to IS (y) against analyte concentration (x). The resulting equations for LB-100 and endothall were obtained over three different days. For LB-100 the equation was Y = 0.01023 (±0.00025) X + 0.007803 (±0.00019); for endothall, it was Y = 0.03160 (±0.00110) X + 0.031467 (±0.00961). The average correlation coefficients (r2), weighted by 1/x2, were found to be 0.995 (0.03 %CV) and 0.999 (0.03 %CV) for LB-100 and endothall, respectively, based on six validation runs. The accuracies and precisions of all calibrators are summarized in Table 5 for LB-100, where accuracy ranged from -9 to 11% and precision ranged from 2 to 6%; and for endothall, where accuracy ranged from -4 to 5% and precision ranged from 1 to 5%. All calibrators (100%) over three different days met the FDA criteria, which demonstrates the performance of the assay.
Table 5. . Accuracy and precision of LB-100 and endothall in pooled human plasma calibrators over three different days.
| Analyte | Nominal concentration (ng/ml) | Mean measured (ng/ml) | SD (ng/ml) | Precision (%CV) | Accuracy (%RE) |
|---|---|---|---|---|---|
| LB-100 | 2.50 | 2.30 | 0.13 | 6 | -8 |
| 5.00 | 5.56 | 0.13 | 2 | 11 | |
| 10.0 | 10.6 | 0.4 | 3 | 6 | |
| 25.0 | 27.0 | 0.6 | 2 | 8 | |
| 50.0 | 52.3 | 1.2 | 2 | 5 | |
| 100 | 94.9 | 2.0 | 2 | -5 | |
| 200 | 183 | 8.0 | 5 | -9 | |
| 500 | 459 | 16.0 | 4 | -8 | |
| Endothall | 2.50 | 2.48 | 0.13 | 5 | -1 |
| 5.00 | 5.16 | 0.18 | 4 | 3 | |
| 10.0 | 9.70 | 0.20 | 2 | -3 | |
| 25.0 | 24.9 | 0.5 | 2 | -1 | |
| 50.0 | 52.4 | 0.6 | 1 | 5 | |
| 100 | 97.2 | 1.5 | 2 | -3 | |
| 200 | 207 | 3.0 | 1 | 3 | |
| 500 | 481 | 14.0 | 3 | -4 |
CV: Coefficient of variation; RE: Relative error; SD: Standard deviation.
Precision & accuracy
As shown in Table 6, the inter-run precision ranged from 2 to 6% for LB-100 and from 2 to 4% for endothall. The inter-run accuracy was within ±9% and ±6% for LB-100 and endothall, respectively (Table 6). The inter-run precision and accuracy were assessed by performing five parallel measurements of five identical QCs at each concentration across three independent validation batches. Intra-assay precision and accuracy were determined through five repeated measurements of each QC sample. Table 7 summarizes the intra-run precision and accuracy values, which were within ±6% for LB-100 and ±9% for endothall, respectively. The intra-run accuracy ranged from -4 to 0% and from -3 to 7% for LB-100 and endothall, respectively.
Table 6. . Inter-run accuracy and precision for LB-100 and endothall in pooled human plasma.
| Analyte | Nominal concentration (ng/ml) | Mean measured (ng/ml) | SD (ng/ml) | Precision (%CV) | Accuracy (%RE) |
|---|---|---|---|---|---|
| LB-100 | 2.50 | 2.41 | 0.14 | 6 | -4 |
| 8.00 | 8.41 | 0.23 | 3 | 7 | |
| 40.0 | 43.4 | 1.0 | 2 | 9 | |
| 400 | 402 | 10.0 | 2 | 0 | |
| Endothall | 2.50 | 2.34 | 0.09 | 4 | -6 |
| 8.00 | 7.99 | 0.21 | 3 | 4 | |
| 40.0 | 40.1 | 0.5 | 1 | 0 | |
| 400 | 413 | 9.0 | 2 | 3 |
Each datum point was calculated by five parallel measurements of five identical quality controls at each concentration over three validation batches.
CV: Coefficient of variation; RE: Relative error; SD: Standard deviation.
Table 7. . Intra-run accuracy and precision for LB-100 and endothall in pooled human plasma.
| Analyte | Nominal concentration (ng/ml) | Mean measured (ng/ml) | SD (ng/ml) | Precision (%CV) | Accuracy (%RE) |
|---|---|---|---|---|---|
| LB-100 | 2.50 | 2.49 | 0.09 | 4 | 0 |
| 7.50 | 7.22 | 0.46 | 6 | -4 | |
| 40.0 | 39.0 | 1.5 | 4 | -3 | |
| 400 | 400 | 15 | 4 | 0 | |
| Endothall | 2.50 | 2.61 | 0.23 | 9 | 5 |
| 7.50 | 8.05 | 0.49 | 6 | 7 | |
| 40.0 | 41.1 | 1.4 | 3 | 3 | |
| 400 | 387 | 11 | 3 | -3 |
Each datum point was calculated by five replicate measurements of each quality control sample within a validation batch.
CV: Coefficient of variation; RE: Relative error; SD: Standard deviation.
Stability
As discussed earlier, LB-100 was found to degrade in acidic conditions; therefore NaOH was added into stock solutions and plasma samples to maintain the stability of LB-100. Under our recommended conditions, a stability study was conducted for LB-100 and endothall, and the results, which are summarized in Table 8, demonstrate the robustness of our methodology. Specifically, at room temperature on the benchtop, both LB-100 and endothall stock solutions were stable over a period of 24 h. Likewise, the stability of LB-100 and endothall in plasma were maintained for at least 24 h. We also investigated the stability of LB-100 and endothall in plasma after sample preparation in the autosampler at 4°C and observed their stability for 24 h. Moreover, the study confirmed no significant impact of repeated freeze–thawing (three cycles) on plasma samples spiked with LB-100 and endothall at low and high QC concentrations. Finally, we assessed the long-term stability of LB-100 and endothall in plasma at -20°C for 60 days, and observed no significant loss.
Table 8. . Stability studies of LB-100 and endothall under various conditions.
| Analyte | Stability recoveries, % (mean ± SD) | ||||
|---|---|---|---|---|---|
| Auto-sampler (4°C, 24 h) | Benchtop (RT, 24 h) | Long-term (-20°C, 60 days) | Freeze–thaw (-20°C after three cycles) | ||
| LB-100 | Plasma LQC | 102 ± 1 | 100 ± 2 | 106 ± 4 | 104 ± 2 |
| Plasma HQC | 97 ± 2 | 104 ± 4 | 92 ± 2 | 92 ± 4 | |
| Endothall | Plasma LQC | 96 ± 1 | 103 ± 5 | 102 ± 5 | 106 ± 6 |
| Plasma HQC | 91 ± 3 | 103 ± 3 | 99 ± 2 | 100 ± 5 | |
HQC: High-concentration quality control; LQC: Low-concentration quality control; RT: Room temperature; SD: Standard deviation.
Application of the method
The validated method was applied to the determination of LB-100 and endothall in plasma samples from an ongoing phase Ib open-label study of LB-100 in untreated extensive-stage small-cell lung carcinoma [18]. Figure 4 shows the LB-100 and endothall concentration–time profile in a patient receiving LB-100 at a dose of 1.25 mg/m2 on days 1 and 3, administered as a 15-min intravenous infusion. The pharmacokinetics of LB-100 displayed similar profiles on days 1 and 3. Circulating plasma concentrations of endothall were low, but detectable by the proposed method, which indicates suitability of the LLOQ of the analytical method for pharmacokinetic studies. Both LB-100 and endothall were also detectable prior to the dose on day 3, suggesting some accumulation (especially for endothall) with multiple doses. The maximal concentration of endothall was observed at the last sampling time point, which precluded the determination of the elimination half-lives for endothall; therefore an extension of the last sampling time point after infusion was recommended for future sampling processes.
Figure 4. . Plasma concentration–time profiles.
LB-100 (solid line and circles) and endothall (dashed line and squares) plasma concentration–time profiles in a patient receiving LB-100 at a dose of 1.25 mg/m2 on days 1 and 3 administered as a 15-min intravenous infusion.
Conclusion
The objective of our study was to address the lack of available analytical methods for the determination of LB-100 and its metabolite, endothall, in human plasma to fill a critical gap in the analytical tools available for therapeutic monitoring of those compounds in clinical setting. Although separate assays have been reported for LB-100 and endothall in biological matrices, there is a lack of a unified method for their simultaneous determination. The difficulties associated with developing such a method are due to the ex vivo stability and differential polarity of LB-100 and endothall. In this study we successfully overcame those challenges, and our study represents the first report of a method for the simultaneous analysis of LB-100 and endothall in human plasma using LC–MS/MS. It has a linear calibration range of 2.50–500 ng/ml for both LB-100 and endothall. The method has been successfully validated and applied to the measurement of LB-100 and endothall in the blood samples of patients. The advantages of the proposed assay, including its high sensitivity (LLOQ: 2.5 ng/ml), low sample volume requirement (100 μl) and short analysis time, are essential for accurate determination of the pharmacokinetics of LB-100 in clinical settings, especially in ongoing and future clinical trials where therapeutic monitoring is crucial.
Executive summary.
An LC–MS/MS method was developed to simultaneously quantify LB-100 and its major metabolite, endothall, in human plasma.
A novel assay was validated according to US FDA guidelines, over a linear range of 2.5–500 ng/ml for both LB-100 and endothall.
A solid-phase extraction was established for sample preparation that effectively removed the interference of endogenous plasma and significantly increased the sensitivity of the assay.
A 6-min LC–MS/MS method was established in multiple reaction monitoring mode with a Hypercard column.
Benchtop stability was established for 24 h, and there was no significant impact of three freeze–thaw cycles on LB-100 and endothall stability in human plasma.
Long-term stability was determined by subjecting plasma samples to storage at -20°C for about 60 days.
The method is being used to successfully quantify LB-100 and endothall in clinical samples obtained from patients recruited to an ongoing phase Ib trial.
Acknowledgments
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Footnotes
Financial & competing interests disclosure
Research reported in this publication includes work performed in the Analytical Pharmacology Shared Resource supported by the National Cancer Institute of the National Institutes of Health under grant no. P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. E Forman and J Kovach are paid employees of the project sponsor and holder of the IND for LB-100, Lixte Biotechnology, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
All patient samples analyzed and reported here were collected following acquisition of informed consent under an Institution Review Board-approved protocol (20068) according to the City of Hope National Medical Center ethical and regulatory guidelines and registered under the clinical trials registry no. NCT04560972.
References
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