Simultaneous Determination and Validation of Oncrasin-266 and its Metabolites by HPLC-MS/MS: Application to a Pharmacokinetic Study

Abstract

Oncrasins are a class of RNA polymerase II inhibitors. Oncrasin-72 is an indole-3 carbinol analog that has shown to inhibit growth and induce the cell death of various human cancer cell lines. Oncrasin-266, a prodrug of oncrasin-72, has been shown to have improved pharmacokinetic properties and safety than Oncrasin-72. With respect to the potential therapeutic advantages of this class of compounds, there is a need for further preclinical assessment for future clinical trials. The development of and validation of an analytical method is essential for the quantification of oncrasins in biological fluids for pharmacokinetic studies. This study focuses on the LC-MS/MS method development and validation of oncrasin-266, oncrasin-72 and its aldehyde metabolite in rat plasma. Blank rat plasma, coupled with 1-(3-chlorobenzyl)-1H-indole, as internal standard, was used for generating standard curves ranging from 1–250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5–125 ng/mL for the aldehyde metabolite. The chromatographic separation was achieved by a Zorbax 300SB-C₁₈ HPLC column at 50°C with a flow rate of 1.1 mL/min under gradient elution. Mass detection was performed under positive ionization electrospray. Intra- and inter-day accuracy and precision of the assay were less than 10%. We report a simple, specific and reproducible LC-MS/MS method for the quantification of oncrasins in rat plasma. This study was successfully used for the quantification of oncrasins in rat plasma for pharmacokinetic studies in three dose groups of 10, 25, and 50 mg/kg via intravenous administration.

1. Introduction

The global burden of cancer on public health is significantly high, with more than 14 million new cases and 8.2 million deaths in 2012 [1]. According to the American Cancer Society, in 2016 there is an expected 1,685,210 cases and 595,690 expected deaths in the US alone [2]. Thus, the discovery and rise of more effective and safe anticancer agents holds prime importance for more efficient management. Our previous studies report oncrasins, a unique class of RNA polymerase II inhibitors [3–6]. Specifically, Oncrasin-72 (NSC-743380) is an indole-3 carbinol analog that has shown to inhibit growth and induce the cell death of various human cell lines derived from lung, colon, breast, ovary, and kidney cancers [7]. In vivo, oncrasin-72 is rapidly metabolized to carboxylic and aldehyde derivatives. Oncrasin-266, a prodrug of oncrasin-72 has shown improved in vivo stability, pharmacokinetic properties, and safety than oncrasin-72 [6]. Oncrasin-266 is converted to oncrasin-72 via ester hydrolysis (in vitro) and further metabolized to its major metabolites carboxylic acid derivative (NSC-751172, inactive) and aldehyde derivative (NSC-741908, active) - represented in Figure 1.

Figure 1.

Chemical structure of oncrasin-266, oncrasin-72, and NSC-741908.

With respect to the potential therapeutic advantages of this class of compounds, there is a need for further preclinical assessment to move the compounds from the product development stage to clinical trials. The development of and validation of an analytical method is essential for the determination of key characteristics, such as the pharmacokinetics, stability and toxicological attributes. Limited pharmacokinetic studies of oncrasins have been reported. Our previous study utilized HPLC-MS for the quantification of oncrasin-266 and oncrasin-72 in mice plasma [6], but the assay was not validated. For two consecutive days, Eldridge, et al. [8] orally administered oncrasin-72 and its two major metabolites to rats. The study provides a preliminary plasma concentration versus time profiles of the analytes but doesn’t provide important pharmacokinetic parameters. Additionally, the study does not provide the reader with detail regarding the method of analysis utilized for determination of plasma drug levels. Therefore, here is a need for a specific and sensitive assay for the quantitative measurement of the analytes in a biological matrix such as rat plasma to evaluate the pharmacokinetics of the analytes [9]. In this study, we developed and validated a simple, sensitive, and specific LC-MS/MS method for the simultaneous determination of oncrasin-266, oncrasin-72, and NSC-741908 in plasma. Subsequently, this validated method was successfully utilized for the quantification of oncrasins in rat plasma for pharmacokinetic studies.

2. Material and Methods

2.1. Materials

Oncrasin-266, Oncrasin-72, NSC-741980, and internal standard (IS, 1-(3-chlorobenzyl)-1H-indole) were synthesized as described previously [5, 6]. These compounds were 96%, 97.5%, 99%, and 99% pure, respectively. Purity and molecular weight was confirmed by HPLC/MS/UV₂₃₀. Formic acid, LC-MS grade acetonitrile and water were purchased from Sigma-Aldrich (St. Louis, MO).

2.2. Preparation of stock solutions

Stock solutions of analytes and IS (500 μg/mL) were prepared by dissolving each substance in acetonitrile, and stored at −20°C until used. A series of standard samples were prepared by diluting the stock solution with acetonitrile and then spiking in blank rat plasma to obtain the following concentrations: 1, 2.5, 5, 10, 25, 50, 100, and 250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5, 1.25, 2.5, 5, 12.5, 25, 50, and 125 ng/mL for NSC-741908. The low, medium, and high concentration levels of quality control (QC) samples were prepared by the same method as the standards at 2, 20, and 200 ng/mL, respectively, for oncrasin-266 and oncrasin-72; and 1, 10, and 100 ng/mL, respectively, for NSC-741908. IS working solution was prepared by diluting the stock solution with acetonitrile to obtain a concentration of 125 ng/mL.

2.3. Sample preparation

Plasma samples stored at −80°C were thawed at room temperature. An aliquot of 50 μL plasma or urine sample was pipetted into a 1.5 mL graduated microcentrifuge tube and spiked with 100 μL internal standard working solution (125 ng/mL). The mixture was centrifuged at 20,800 x g for 5 min at 4°C. The supernatant was transferred into the polyethylene autosampler vial and injected into the LC–MS/MS for analysis.

2.4. Instrument conditions

HPLC analysis was carried out with a Shimadzu Nexera HPLC equipped with a Zorbax 300SB-C₁₈ HPLC column (50 x 4.6 mm, 3.5μm) at 50°C with a flow rate of 1.1 mL/min. Each sample injection volume was 5 μL and the mobile phase consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B). Gradient elution was employed with 60–95% B (0–1.8 min) and kept constant at 95% B for 1.2 min, 95–60% B (3–3.5 min) and kept constant at 60% B for 1.5 min.

MS/MS analysis was carried out on 4000 Q Trap triple quadrupole LC-MS/MS system with a Turbo Ion Spray ion source (Applied Biosystem/MDS Sciex). The quantification for each analyte was performed in multiple-ion reaction monitoring (MRM) mode operated in positive, electrospray ionization (ESI) mode. The source parameters were set as follows: ion spray voltage, 5500 V; ion source temperature, 650°C; nebulizer gas, 50 psi; heater gas, 65 psi; curtain gas, 15 psi; and the collision gas, high. The LC-MS/MS system was controlled and data was acquired by Analyst software version 1.6.2. The compound dependent parameters were optimized with entrance potential (EP) at 10V, and dwell time at 150 ms for all compounds. The remainder compound-dependent parameters are summarized in Table 1.

Table 1.

Compound dependent parameters for Oncrasins and IS in MRM mode for LC-MS/MS analysis.

Analyte	[M+H]+	MRM transition	DP (V)	CE (V)	CXP (V)
Oncrasin-266	255.1	125	56	39	10
Oncrasin-72	255.1	125	90	31	7
NSC-741908	270.2	125	60	18	13
IS	242.0	125	52	31	11

2.5. Validation

The LC-MS/MS assay described herein has been validated according to Center for Drug Evaluation and Research (CDER) “Guidance for Industry: Bioanalytical Method Validation”[9].

2.5.1. Calibration curve

Calibration curves in blank rat plasma were created by plotting the peak area ratio of each analyte to IS versus the known concentrations of each compound.

2.5.2. Extraction recovery and matrix effect

To examine the extraction recovery and matrix effect, the QC samples at three concentration levels were evaluated. The extraction recovery and matrix effect were calculated according to Eq. 1 and Eq. 2 respectfully.

$$\%\text{Recovery}=\frac{{\text{Response}}_{\text{pre}-\text{extraction}\text{spike}}}{{\text{Response}}_{\text{post}-\text{extraction}\text{spike}}}\times 100$$ (Eq. 1)

$$\%\text{Matrix}\text{factor}=\left(\frac{{\text{Response}}_{\text{post}-\text{extraction}\text{spike}}}{{\text{Response}}_{\text{matrix}-\text{free}\text{sike}}}\right)\times 100$$ (Eq. 2)

Where Response_{pre-extraction spike} is the mean peak area count for oncrasin samples that have undergone the extraction process. Response_{post-extraction spike} is the mean peak area count for oncrasin samples spiked into extracted matrix after the extraction procedure. Response_{matrix-free spike} is the mean peak area count for oncrasin samples at the same concentration in water. The RSD of recovery and matrix effect at each concentration should not exceed 15%. Experiments were conducted in triplicate.

2.5.3. Accuracy and precision

The intra- or inter-day accuracy and precision of the LC-MS/MS method were evaluated by analyzing the QC samples via calibration curves created on the same day or three different days. Experiments were conducted in sextuplicate.

2.5.4. Stability

All stability studies were evaluated in blank rat plasma at low and high QC levels (2 and 200 ng/mL, respectively) using three replicates at each concentration level. All the samples were compared to freshly prepared samples at the same concentrations. QC samples in blank rat plasma were freshly prepared and left on the bench-top at room temperature for 1, 2 and 3 h (short-term benchtop). Freeze-thaw (FT) stability samples were exposed to three cycles of freeze (−80°C) and thaw (RT, room temperature). The stability of processed sample was determined by the comparison of freshly obtained plasma extracts to plasma extracts that remained in the auto-sampler for 1 h and 24 h at 15°C. Long-term storage stability samples were freshly prepared and stored at 80°C for 14 days.

2.6. Plasma protein binding

Plasma protein binding (PPB) studies were conducted using a modified ultra-filtration technique [10]. Briefly, stock solutions were diluted with acetonitrile and spiked in blank rat plasma at three different concentrations: 50, 100, and 500 ng/mL for oncrasin-266 and oncrasin-72; and 25, 50, and 250 ng/mL for NSC-741908. The plasma was incubated at 37 °C for 10 min before being transferred to Amicon Ultra-0.5 mL centrifugal filters of 30kDa (EMD Millipore Corporation, Billerica, MA) for ultrafiltration at 20,800 x g for 15 min at 4°C. Filtrate and nonfiltrate plasma concentrations were spiked with IS and analyzed by LC-MS/MS.

Drug protein binding was calculated as PB = [1 ( Cu/Cp)]. Where PB represents protein binding, Cu is the unbound drug concentration, and Cp is the protein-bound drug concentration. Experiments were conducted in triplicate.

2.7. Assay application in pharmacokinetic study

This validated LC-MS/MS method was successfully applied to a pharmacokinetic study involving quantification of oncrasins in rat plasma and urine. The pharmacokinetic study in rats was performed using the protocol approved by Texas Southern University Animal Care and Use Committee. The oncrasin-266 prodrug formulation was administered to healthy male adult Sprague-Dawley rats at a dose of 10 mg/kg (n=5), 25 mg/kg (n=5) and 50 mg/kg (n=5) via intravenous injection through jugular vein cannula.

Blood samples of 200μL were taken before dose administration (0 hour), and at 0.0167, 0.0833, 0.25, 0.5, 1, 2, 3, 5, 7, 10, 24, 32, 48 hours post dosing and placed into heparinized microfuge tubes. Plasma was harvested after centrifugation of blood at 20,800 x g for 3 min and stored at −80°C until analysis. Urine samples were collected from 0 to 24 hours and stored at −80°C until analysis. The plasma concentration versus time profiles were analyzed for each rat using a noncompartmental model (WinNonlin v6.4, Pharsight Corp, Mountain View, CA).

2.8. Statistical analysis

Data for C_max, and AUC_last were analyzed nonparametrically to study dose proportionality. All tests were dose-normalized, utilized a 0.05 level of significance and used the Kruskal-Wallis multiple comparisons test for each dose comparison.

3. Results and discussion

3.1. Chromatographic and mass spectrometry optimization

Several mobile phase compositions and columns were tested. It is recommended to use a stable isotope-labeled analog as internal standard for quantitative LC–MS/MS analysis [11] and [12]. The best peak response was attained with acetonitrile and water with 0.1% FA as opposed to 0% and 0.2% FA. It was found that the Zorbax 300SB-C₁₈ column (50 x 4.6 mm, 3.5μm) achieved optimal peak shape, response, and shortest retention time, compared to a Zorbax Eclips Plus C₁₈ (50 x 4.6mm, 3.5 μm), Xterra MS C₁₈ (50 x 2.1mm, 3.5μm), and Poroshell 120EC C₁₈ (50 x 2.5mm, 2.7μm) column. Other parameters such as column temperature (45–50°C and RT), gradient optimization, flow rate (0.4–1.2 mL/min), and injection volume were studied. The Zorbax 300SB-C₁₈ column along with column temperature of 50°C, flow rate of 1.1 mL/min was found to be most suitable. Positive ion mode and ESI mode was found to provide the best peak intensity, versus negative mode and atmospheric chemical ionization (APCI) mode. After collision cell fragmentation, the most abundant MRM transition was 125 for all analytes. Optimization of source parameters was studied to determine the most suitable conditions for the analytes.

3.2. Method validation

3.2.1. Linearity and carryover

The linearity of the calibration curves was established over the concentration range of 1 – 250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5–125 ng/mL for NSC-741908. The correlation coefficient values >0.999 for all validated batches. No peaks near same retention time of oncrasin-226, oncrasin-72, and NSC-741908 were detected in blank plasma chromatograms that were injected immediately after injection of the highest calibration standard. Therefore, carryover from samples was determined to be negligible.

3.2.2. Extraction recovery and matrix effect

The mean extraction recovery for oncrasin-266, oncrasin-72 and NSC-741908 were 83.35%, 96.50%, and 93.99%, respectfully, at the low QC concentration, 83.83%, 103.58% and 93.96% at the medium QC concentration, and 80.44%, 100.36%, and 89.58% at the high QC concentration.

Matrix effect was measured to determine potential ion enhancement or suppression caused by co-eluting matrix components. The matrix factors for oncrasin-266, oncrasin-72 and NSC-741908 were 101.55%, 95.12% and 101.12%, respectively, at the low QC concentration, 81.06%, 96.20%, and 104.00% at the medium QC concentration, and 80.39%, 96.23% and 97.86% at the high QC concentration, No significant matrix effect was considered if RSD was within ±15%. The RSD of matrix factors was equal to or less than 12%, suggesting no significant matrix effect for all analytes. This data shows that the simple sample preparation method yielded very high and stable extraction recovery. Also, the sample preparation method plus the use of Zorbax 300SB-C₁₈ HPLC column resulted in no measurable matrix effect.

3.2.3. Accuracy and precision

The intra- and inter-day accuracy (present as relative error) and precision (present as coefficient of variation) of the assay were less than 10% (results are summarized in Table 2). This data indicates that the developed LC-MS/MS method is accurate and precise for the analysis of prodrug oncrasin-266 and its metabolites in plasma samples at a concentration of 1 - 250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5–125 ng/mL for NSC-741908.

Table 2.

Intra- and inter-day accuracy and precision of oncrasins

Oncrasin-266	Intra-day (n=6)		Inter-day (n = 6)
	Accuracy (RE*, %)	Precision (CV*, %)	Accuracy (RE, %)	Precision (CV, %)
Low	1.83	2.30	2.67	5.74
Med	6.42	4.35	0.56	6.11
High	5.75	2.24	1.68	5.59
Oncrasin-72	Intra-day (n=6)		Inter-day (n = 6)
	Accuracy (RE*, %)	Precision (CV*, %)	Accuracy (RE, %)	Precision (CV, %)
Low	2.02	1.85	0.52	2.86
Med	0.08	1.98	0.55	3.12
High	0.82	0.38	1.06	2.15
NSC-741908	Intra-day (n=6)		Inter-day (n = 6)
	Accuracy (RE*, %)	Precision (CV*, %)	Accuracy (RE, %)	Precision (CV, %)
Low	2.09	4.5	1.53	9.45
Med	2.38	4.70	0.32	7.67
High	7.14	3.30	0.60	6.51

3.2.4. Stability

Stability studies were conducted to evaluate the stability of the analytes under expected sample handling and storage conditions. The results of the stability study are expressed as the mean remaining percentages of nominal concentration (Table 3). The analytes were found to be stable up to 1 h at room temperature. However, oncrasin-266 degraded by an additional 18% after 2 h at room temperature, while the oncrasin-72 and NSC-741908 remained stable (data not shown). The data shows that the compounds are stable in rat plasma at room temperature for at least 1 h. Such a short sample preparation time is sometimes challenging. Oncrasin-266 is unstable after 1 h because of the high levels of esterase present in rodent blood. Therefore, the utilization of an esterase inhibitor is advised for future studies to ensure the stability of the prodrug during sample handling [13].

Table 3.

Stability of oncrasins in rat plasma expressed as percent of nominal concentration [n=3; mean (±SD)].

Mean ± S.D. (%)
Analyte	Nominal concentration (ng/mL)	Short-term (1h)	Three freeze-thaw cycles	Auto-sampler at 15°C (1h)	Long term -80°C (14 days)
Oncrasin-266	2	99.05 ± 12.00	100.88 ± 8.73	94.46 ± 6.16	99.01 ± 1.11
	200	85.67 ± 4.16	86.88 ± 4.56	91.34 ± 7.10	100.35 ± 1.63
Oncrasin-72	2	94.87 ± 5.31	97.53 ± 5.94	94.90 ± 2.54	95.02 ± 8.54
	200	110.55 ± 3.0	101.23 ± 10.03	98.05 ± 6.15	89.14 ± 0.49
NSC-741908	1	101.00 ± 12.58	110.20 ± 6.79	113.44 ± 6.90	112.67 ± 2.45
	100	99.21 ± 5.62	95.15 ± 4.53	100.25 ± 2.67	93.46 ± 5.62

The freeze-thaw stability of the analytes was within 15% of nominal concentrations which suggested that the compounds in rat plasma were stable after three freeze-thaw cycles. The plasma extracts that remained in the auto-sampler for 1 h at 15°C displayed were within 15% of nominal concentration. However, after 24 h in the auto-sampler, oncrasin-266 displayed an average of only 30.60% of the nominal concentration, while oncrasin-72 and NSC-741908 were within the 15% of nominal concentration (data not shown). This indicates that only parent and metabolite are stable after 24 h in the auto-sampler at 15°C. Long-term storage stability samples values were within 15% of nominal concentrations, indicating that the pharmacokinetic samples could be stored up to two weeks at 80 °C without degrading the integrity of the sample.

3.3. Plasma Protein Binding

The plasma protein binding of oncrasin-266 and its metabolites were determined by a modified ultrafiltration method [10]. The plasma protein binding for oncrasin-266, oncrasin-72, and NSC-741908 were 99.5 ± 0.81%, 99.0 ± 0.76%, and 99.2 ± 0.63%, respectively. There was no difference in binding across concentrations tested (50 – 500 ng/mL).

3.4. Pharmacokinetic studies

Figure 2 shows representative chromatograms of (a) blank rat plasma spiked with IS; (b) blank rat plasma spiked with 250ng/mL of oncrasin-266 and oncrasin-72, 125ng/mL of NSC-741908, and IS; and (c) rat plasma sample from pharmacokinetic study, with calculated concentration of 187.58 ng/mL for oncrasin-266 and 32.09 ng/mL for oncrasin-72. Figure 3 shows plasma concentrations versus time profiles of oncrasin-266, oncrasin-72, and NSC-741908, respectively, following intravenous administrations of oncrasin-266. All three compounds showed bi-exponential disposition with initial rapid distribution of each compound followed by a slow elimination process. Pharmacokinetic parameters of each compound following three dose levels are shown in Table 4. Estimated mean maximum concentrations of oncrasin-266 were 61.1 μg/mL, 86.2 μg/mL, and 238.1 μg/mL after intravenous administration of 10 mg/kg, 25 mg/kg and 50 mg/kg of oncrasin-266, respectively. The prodrug was immediately hydrolyzed to the active drug oncrasin-72. The metabolic process of the conversion seems maximized at the dose level of 25 mg/kg with mean C_max of oncrasin-72 reaching 52.4 μg/mL, while at the dose level of 50 mg/kg the mean C_max of oncrasin-72 did not change accordingly (54.8 μg/mL). It is interesting to know that oncrasin-266 and oncrasin-72 had very similar terminal elimination half-lives, suggesting elimination-rate limiting process in vivo. The active aldehyde metabolite had much lower concentration and faster elimination as compared to the parent compound oncrasin-72.

Figure 2.

Mass chromatograms of (a) blank rat plasma spiked with IS; (b) blank rat plasma spiked with 250ng/mL of oncrasin-266 and oncrasin-72, 125ng/mL of NSC-741908, and IS; and (b) rat plasma sample from pharmacokinetic study, with calculated concentration of 187.58 ng/mL for oncrasin-266 and 32.09 ng/mL for oncrasin-72.

Figure 3.

Semi-log plot of the mean plasma concentration-time curves for (a) oncrasin-266, (b) oncrasin-72, and (c) NSC741908 after I.V. administration of the oncrasin-266 formulation at 10, 25, and 50 mg/kg (mean ± SEM).

Table 4.

Pharmacokinetic parameters (mean ± SD) of oncrasin-266, oncrasin-72, and NSC-741908 at dose 10, 25, and 50 mg/kg after I.V. dose of the oncrasin-266 formulation.

Analyte	Dose (mg/kg)	Cmax (ug/mL)	AUC (h·μg/mL)	Vd (L/kg)	Cl (L/kg)	t1/2 (h)
Oncrasin-266	10	61.05 ± 11.79	4.37 ± 0.75	26.44 ± 14.25	2.34 ± 0.37	7.58 ± 3.58
	25	86.23 ± 30.2	8.03 ± 2.61	41.27 ± 24.19	3.39 ± 1.17	7.93 ± 2.77
	50	238.12 ± 121.46	70.18 ± 28.2	8.11 ± 2.49	0.79 ± 0.25	7.13 ± 0.90
Oncrasin-72	10	15.55 ± 5.33	2.22 ± 0.33	45.06 ± 16.70	4.56 ± 0.7	7.01 ± 2.81
	25	52.44 ± 16.75	7.15 ± 1.96	42.55 ± 24.3	3.62 ± 0.77	7.74 ± 3.95
	50	54.79 ± 30.6	23.70 ±7.95	27.78 ± 10.36	2.30 ± 0.74	8.28 ± 1.34
NSC-741908	10	1.56 ± 1.55	0.50 ± 0.42	47.04 ± 33.52	75.73 ± 86.14	1.19 ± 0.93
	25	0.33 ± 0.12	0.11 ± 0.03	443.15 ± 71.91	254.62 ± 108.12	1.31 ± 0.39
	50	2.14 ± 1.73	0.68 ± 0.42	137.64 ± 33.49	103.60 ± 67.32	1.16 ± 0.52

Mean AUC of oncrasin-266 increased proportionally from 4.37 to 7.15 h·μg/mL when doses were increased from 10 mg/kg to 25 mg/kg. However, when the dose was increased to 50 mg/kg, AUC had a disproportional drastic increase to 70.2 h·μg/mL. The data suggest pharmacokinetics of oncrasin-266 is linear under doses of 25 mg/kg, but displays nonlinear pharmacokinetic behavior at doses of 50 mg/kg. This was probably due to saturation of the hydrolysis process of the prodrug oncrasin-266 at a higher dose. A similar phenomena was observed for oncrasin-72.

The cumulative amount of unchanged compounds excreted in the urine was found to be <0.02% for all the compounds, suggesting renal elimination is negligent and the compounds were exclusively metabolized in the liver.

4. Conclusion

We have developed and validated a simple, specific, and reproducible LC-MS/MS method for the quantification of oncrasin-266, oncrasin-72, and NSC-741908 in rat. This method was validated to be accurate and precise over the concentration range of 1–250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5 – 125 ng/mL for NSC-741908. This study provides preliminary evidence that prodrug oncrasin-266 pharmacokinetic parameters increase proportionally with dose at 10 – 25mg/kg, but display nonlinear pharmacokinetic behavior at doses 50 mg/kg. Our identification of the clearance and dose-linearity of oncrasin-266 may help determine future dosing recommendations of the compound.

Highlights.

• A validated, simple, and reproducible LC-MS/MS method for the quantification of oncrasin-266, oncrasin-72, and NSC-741908 (a class of RNA polymerase II inhibitors- potential anti-cancer agent).

• The method is suitable for the quantification of oncrasins in rat plasma for pharmacokinetic studies.

• Method was validated to be accurate and precise over the concentration range of 1–250 ng/mL for oncrasin-266 and oncrasin-72; and 0.5–125 ng/mL for NSC-741908.

• Results suggest that the pharmacokinetics of oncrasin-266 is linear under doses of 25 mg/kg.