MEDICINAL CHEMISTRY RESEARCH

Med Chem Res (2015) 24:459–467 DOI 10.1007/s00044-014-1106-x

ORIGINAL RESEARCH

Rapid determination of lenalidomide in rat plasma by an ultra performance liquid chromatography/tandem mass spectrometric method Krishna Pocha • G. Devala Rao

Received: 24 December 2013 / Accepted: 12 June 2014 / Published online: 16 July 2014 Ó Springer Science+Business Media New York 2014

Abstract A new, rapid, and specific ultra performance liquid chromatographic assay with mass spectrometric detection has been developed and validated for the quantitative determination of the antiangiogenic agent lenalidomide in rat plasma using thalidomide as an internal standard with electro spray ionization in multiple-reaction monitoring mode. The analyte and internal standard were extracted by liquid–liquid extraction using methyl-tert-butyl ether solvent. Chromatography was performed isocratically on Acquity BEH C18 column using a mobile phase consisting of a mixture of buffer, acetonitrile, and formic acid in ratios of 30:70:0.1 (v/v/v). The method had a chromatographic run time of 1.2 min. The calibration curve was fit to a linear response concentration data over a range of 0.06–60.00 lM using a weighting factor of 1/92. The intra- and inter-day precision and accuracy results were well within the acceptable limits. The validated method was successfully applied for routine analysis of the pharmacokinetics of lenalidomide. Keywords Lenalidomide
Ultra performance liquid chromatography
Tandem mass spectrometry
Low sample volume
Pharmacokinetics
High throughput analysis

Introduction Lenalidomide (LLM) is a novel oral immunomodulatory drug (Fig. 1), with antiangiogenic and antineoplastic

K. Pocha (&)
G. Devala Rao KVSR Siddhartha College of Pharmaceutical Sciences, Pinnamaneni Polyclinic Road, Vijayawada 520010, Andhra Pradesh, India e-mail: [email protected]

properties (Kastritis and Dimopoulos, 2007). It is a synthetic derivative of glutamic acid and is structurally close to thalidomide (TLM) (Fig. 2) but has an improved toxicity profile and more potent immunomodulatory activity (Mitsiades and Mitsiades, 2004). LLM is currently being used in the treatment of hematological malignancies, particularly multiple myeloma, and a subset of myelodysplastic syndrome harboring the deletion 5q chromosome abnormality (Rajkumar et al., 2005; Richardson et al., 2006; List et al., 2005, 2006). It has shown promise in phase II studies for chronic lymphocytic leukemia, nonHodgkin’s lymphoma, amyloidosis, and myelofibrosis with myeloid metaplasia (Chanan-Khan et al., 2006; Wiernik et al., 2008; Witzig et al., 2007; Vaishali et al., 2007; Dispenzieri et al., 2007; Ayalew et al., 2006). Immunomodulatory properties of LLM include the inhibition of cytokines such as VEGF, bFGF, IL-6, and TNF-a. These cytokines are key regulators of tumor growth and angiogenesis (Robert and Judah, 2002). In a recent phase III study, the use of LLM for advanced and refractory multiple myeloma has been associated with significant biological responses in M-protein (Barlogie et al., 2004). In support of further clinical and preclinical pharmacologic studies with LLM, we describe here the development and validation of a specific, accurate, precise, and sensitive analytical method by ultra performance liquid chromatography with mass spectrometry detection for the determination of LLM in rat plasma. In quantitative bioanalysis, the purpose of an internal standard (IS) is meant to correct for variability in sample preparation, stability, chromatography, ionization process, and other instrument parameters. Therefore, IS of LC/MS assays is either structurally analogs or stable isotopically labeled are considered essential in quantitative bioanalytical assays because it exactly mimics the analyte and

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Results and discussion Detection and chromatography

Fig. 1 Lenalidomide: 3-(4-amino-1-oxo-3H-isoindol-2-yl) piperidine2,6-dione

Fig. 2 Thalidomide: 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3 (2H)dione

provides accurate estimation. Literature survey reveals that several methods have been reported for the quantitative determination of LLM using unusual IS with long chromatographic run time and high sample volume. Tohnya et al. (2004) detailed an LC–MS method for the quantification of LLM using umbelliferone as an IS with a run time of 8 min and a lower limit of quantitation (LLOQ) of 5 ng/ mL using 600lL of sample volume. (Nianhang et al., 2012) was published to estimate in vitro samples by LC–MS/MS using 13C5-LLM as the IS, chromatographic separation was achieved by high-performance liquid chromatography (HPLC) with a run time of 5.5 min and a LLOQ of 5 ng/ mL. However, SIL-IS are not routinely available. In this paper, we demonstrate with reproducible results that chromatographically co-eluting structurally analog IS may be well suited for this purpose. This method employs a simple isocratic separation, and a straightforward liquid– liquid extraction (LLE). When compared to the other methods published, this method is more sensitive, and only a low sample volume (25 lL) of plasma is required for the accurate quantification of LLM with shorter run time. Overall, a sensitive straight-forward method has been developed which superior against published literature.

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In order to find most sensitive ionization mode for the LLM studied, ESI positive- and negative-ion mode were tested with various combination of MP, i.e., ACN and ammonium formate buffer (10 mM)/formic acid (0.1 %) in positive- and negative-ionization mode. It was observed that the signal intensity for [M?H]? ions in ESI positive mode was 2–10 fold higher for component in analyses using ACN: ammonium formate buffer/formic acid, versus experiments run with ESI negative-ion mode. The protonated molecular ions of [M?H]? were m/z 260.2 ? 149.1 and 259.2 ? 149.1 for LLM and TLM, respectively. No significant solvent adducts ions or fragment ions were observed in the full scan spectra. Thus, it was decided to utilize positive-ion mode for detection and quantization of [M?H]? ions, which on fragmentation gave prominent and stable product ions. The optimized declustering potential (DP) for protonated [M?H]? of LLM and TLM found to be 52 and 48, respectively. Acetonitrile (ACN) rather than methanol was chosen as the organic modifier because of its better peak shape. Moderately high acidic ammonium formate buffer 10 mM was required to achieve acceptable peak width and shapes. Acquity BEH C18 column (50 9 2.1 mm, i.d. 1.7 lm) was found to be necessary to reduce retention time and thus deliver good peak shape. Final MRM transitions were selected on the basis of signal-to-noise ratio (S/N) with on-column injection analysis. GS1, GS2, Curtain gas (CUR), collision gas (CAD), ion spray voltage (ISV), and temperature were set to 45, 50, 30, medium, 5,500 kV, and 550 °C, respectively. The transitions selected were m/z 260.2 [ 149.1 and 259.3 [ 149.1 for LLM and TLM, respectively. Typical chromatograms resulting from the LC–MS/MS analysis of extracts of 25 lL plasma from a control rat blank sample (Fig. 3), a control rat plasma sample spiked to contain LLM at a LLOQ concentration of 0.06 lM (Fig. 4), ULOQ concentration of 60.00 lM (Fig. 5), and a sample from a rat obtained 1 h after administration of LLM (Fig. 6) is demonstrated the specificity and selectivity of the method. As shown in Figs. 3 and 4, no significant interference in the blank plasma traces was seen from endogenous substances in drug-free plasma at retention time of the analyte and IS. LLM and the IS peaks were well resolved under the optimized conditions. TLM was chosen for quantification as the IS due to its similarity with the analyte in structure, chromatographic, and mass spectrographic behavior, LLM is commercially available and can be quantitated using the assay developed for LLM. The retention times of LLM and the IS

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Fig. 3 Representative chromatogram of blank rat K2 EDTA plasma sample

Fig. 4 Representative chromatogram of the LOQ of quantitation sample

were 0.58 and 0.69 min, respectively. The total sample run time was set to 1.2 min. LLE was advantageous because this technique not only extracted the analyte and IS with sufficient efficiency and specificity, but also minimizes the experimental cost. Ethyl acetate, diethyl ether, ACN, 1-chlorobutane, and n-butanol were all tested as extraction solvent, and MtBE was finally adopted because of its extraction efficiency.

Validation Specificity The specificity of the method was evaluated by analyzing blank plasma samples from six rats. Each blank sample was tested for interference using the proposed extraction procedure and chromatographic/mass spectroscopic conditions

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Fig. 5 Representative chromatogram of a high quality control sample

Fig. 6 Representative chromatogram of the 1 h post dose

and compared with those obtained with an aqueous solution of the analyte at a concentration near the LLOQ. The matrix effect on the ionization of the analyte was evaluated by comparing the peak areas of the analyte resolved in the blank sample (the final solution of blank plasma after extraction and reconstitution) with that resolved in the MP. Three different concentration levels of LLM (0.06, 25.00, and 50.00 lM) and 25 lM of the IS were evaluated by analyzing six samples at each level. The blank plasmas

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used in this study were six different batches of blank rat plasmas. If the ratio is \85 % or [115 %, an exogenous matrix effect is implied. Precision and Accuracy The intra-day precision (expressed by coefficient of variation of replicate analyses) was estimated on the three quality control (QC) levels and the inter-day precision on

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Table 1 Summary of precision and accuracy from QC samples Spiked concentration (lM)

Intra-day (n = 6)

Inter-day (n = 6)

Measured concentration (lM) (mean ± SD)

0.15

R.S.D

Nominal (%)

Measured concentration (lM) (mean ± SD)

R.S.D

Nominal (%)

0.15 ± 0.01

8.47

99.23

0.15 ± 0.01

6.21

98.42

25

26.75 ± 0.58

2.17

106.98

24.81 ± 2.01

8.11

99.26

50

50.21 ± 1.50

2.88

100.41

47.91 ± 2.16

2.15

95.81

Table 2 Extraction recovery in rat plasma (n = 6)

Table 3 Calibration linearity results of LLM

Sample type

Concentration (lM)

Recovery (%)

R.S.D

LLM

0.15

65.14

3.27

25.00

59.18

TLM

Analytical run ID

Y-intercept

Slope

Determination coefficient (r)

3.64

1

0.00401

0.0354

0.9966

2 3

0.00258 0.00105

0.0242 0.0306

0.9980 0.9973

50.00

60.81

2.27

25.00

55.17

6.07

the eight calibration standard levels. Table 1 shows the results obtained for the intra-assay (variation intra-day) and inter-assay (variation inter-day) precision for LLM. The precision for LLM under investigation was not exceeded 15 % at any of the concentrations studied and well met the requirements of validation. Recovery The recovery of LLM and TLM from plasma was estimated at their respective low, medium, and high QC levels. Plasma samples (in six replicates) containing analyte at QC concentration level were also spiked with IS at the working concentration of 25 lM. The samples were subsequently processed using the procedure described previously. A second set of plasma samples was processed and spiked postextraction with the same concentrations of the analytes and IS that actually existed in the pre-extraction spiked samples. Extraction recovery values for each analyte and IS were determined by calculating the ratios of the raw peak areas of the samples spiked after extraction. The results are indicated in Table 2. Calibration curve Limit of detection (LOD) and LLOQ Two criteria were used to define LLOQ: (1) the analytical response at LLOQ must be five times the baseline noise and (2) the analytical response at LLOQ can be detected with sufficient precision (15–20 %) and accuracy (80–120 %). LOD is defined as the lowest concentration of the analyte at which the signal is larger than three times the baseline noise.

4

0.000843

0.0365

0.9973

N

4

4

4

MIN

0.0008

0.0242

0.9966

MAX

0.0040

0.0365

0.9980

The measured LLOQ and LOD values were 20 and 5 arbitrary units for LLM. These results well met the requirements of quantifications of all analytes in plasma. The LOQ was determined to be 0.06 lM. Preliminary pharmacokinetic analysis of clinical specimens indicated that plasma concentrations of LLM were always higher than 0.06 lM at the doses and sampling times specified in the study protocol. Response function For each analytical run, an eight-point plasma standard curve was constructed, and the peak area ratios of analyte to IS in rat plasma were linear over the concentration range for LLM 0.06–60.00 lM. The calibration model was selected based on the analysis of the data by linear regression with and without intercepts (y = mx ? c and y = mx) and weighting factors (1/x, 1/x2 and 1/log x). The model with the least total bias across the concentration range was investigated to obtain the reciprocal of the concentration (1/x2) as the weighting factor. For each calibration curve, the calibrators were back calculated from the response factor and the intercept. The correlation coefficients (R) for all components were above 0.995 over the concentration range used. Table 3 shows the results obtained for summary parameters of linearity for LLM. Stability QC samples were subjected to short-term and long-term storage condition (-70 °C), freeze–thaw stability, bench

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Table 4 Stability in rat plasma (n = 6) Nominal concentration (lM)

Sample condition Bench top stability Nominal (%)

R.S.D

Auto-sampler stability

Freeze–thaw stability

30 days storage stability

Nominal (%)

R.S.D

Nominal (%)

R.S.D

Nominal (%)

R.S.D

0.15

94.00

7.23

99.78

4.32

99.44

10.06

97.22

4.41

50

95.77

8.69

95.50

8.02

96.72

8.04

102.05

8.17

Sample dilution To demonstrate the ability to dilute and analyze samples containing analyte at concentration above the assay upper limit of quantification, a set of plasma samples was prepared containing LLM at a concentration of 180.00 lM and placed in a -70 °C freezer overnight prior to analysis. After thawing, certain aliquot was diluted either with 2 and 4 or 4 and 8 times SD rat blank plasma and analyzed. The results of this experiment indicated that the dilution integrity of all the plasma samples was found to be less than 15 % of their respective nominal concentrations.

100 MPK

LLM-PK Profile

Concentration (µM)

top and auto-sampler stability. All stability studies were carried out at two concentration levels (low and high QC) in six replicates. The bench top stability was studied for low and high QC samples kept at room temperature for 6 h, freeze–thaw stability of low and high QC samples was evaluated after third freeze thaw cycles. After the third freeze–thaw cycle, LLM plasma concentrations had deviations from the nominal values within recommended guidelines, irrespective of the tested plasma concentrations. The auto-sampler stability was studied for low and high QC samples kept at auto-sampler 10 °C for 24 h. The freezer storage stability of the drug in plasma was determined by comparing the low and high QC samples stored for 30 days at -70 °C. The percentage stability was estimated by comparing the mean of back-calculated concentration of all analytes from the stored stability samples with that of freshly spiked QC samples. The results indicated that each analyte had an acceptable stability under those conditions, as shown in Table 4.

50 MPK 10 MPK

50.00 40.00 30.00 20.00 10.00 0.00 0.00

2.00

4.00

6.00

8.00

10.00

Time (h)

Fig. 7 Mean plasma levels of LLM obtained in SD rats (n = 3) after administration of oral doses of 10, 50, and 100 mg/kg

Table 5 Mean (SD) pharmacokinetic parameters of lenalidomide PO Mean ± SD Dose (mg/kg) Cmax (nM) Tmax (h) AUClast (nM*h) thalf (h)

100 38.27 ± 3.62 2±0 325.80 ± 26.15 13.49 ± 2.47

Mean ± SD 50 15.67 ± 0.57 1±0

Mean ± SD 10 4.37 ± 0.81 1±0

108.78 ± 1.45

23.51 ± 5.37

3.90 ± 2.13

1.24 ± 0.41

(AUClast; Nm*h) was 325.80, 108.78, and 23.51 Nm*h for 10, 50, and 100 mg/kg doses, respectively, and the terminal half-life (t1/2) was 2.0 for 100 mg/kg and 1.0 h for 50, 10 mg/kg doses. The dose was linear for 10 and 50 mg/kg, but 100 mg/kg was not in linear, as shown in Table 5. Experimental

Application

Chemicals and reagents

After completion of the validation process, the assay was applied to determine LLM plasma concentrations in a different dose. Concentration–time data for LLM in plasma from after administrating drug orally at a dose of 10, 50 and 100 mg/kg are presented in Fig. 7. Plasma concentration data of LLM were analyzed by non-compartmental methods using the Kinetica software. The maximum peak concentration for LLM was 4.37, 15.67, and 38.27 lM, and the area under the curve extrapolated to infinity

LLM (99.5 % purity) and TLM provided by Natco Pharmaceuticals ltd, Hyderabad, India. ACN and methanol was procured from Qualigens, Mumbai, India. Methyl tertbutyl ether (MtBE) and ammonium formate were purchased from Merck, Mumbai, India, and all other reagents and chemicals are analytical grade. Deionized water was produced from Millipore (U.S.A). Blank rat plasma obtained by centrifugation of blood treated with anticoagulant ethylenediaminetetraacetic acid dipotassium salt

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The mass spectrometer (Applied Bio systems, Model API 4000 with Acquity UPLC Waters Corp, USA) equipped with turbo ion spray source using positive ionization mode [Es?] using in multiple reaction monitoring (MRM) and analysis were performed using the analyst software 1.4.2. An Acquity BEH C18 column (50 9 2.1 mm, i.d. 1.7 lm (Milford, MA, USA)) was used for analyte separation. The mobile phase (MP) consisted of 10 mM ammonium formate: ACN and formic acid in ratios of 30:70:0.1 (v/v/v). The MP was delivered isocratically at a flow rate of 0.5 mL/min. The analytical column was kept at 40 °C. The sample injection volume was 5 lL. Total sample run time was 1.2 min. The MS system was operated in the positiveion mode, and the conditions were optimized to generate maximum analyte signal. The MS conditions were as follows: Ionization mode: Electrospray ionization (turbo ionspray), positive, Scan type: MRM, CUR: 30 psi, CAD: medium, Ion source gas (Gas 1): 45 psi, ion source gas (Gas 2): 50 psi, ISV: 5500 V, Temperature: 550 °C. MRM was accomplished at m/z 260.2/149.1 for LLM and m/z 259.3/149.1 for the IS, TLM, as protonated molecular ions. Both ions had a dwell time of 100 ms and were monitored in the unit-resolution mode.

analytes in rat plasma. Hence the precipitation method was tried initially with ACN (0.5 mL) and it has shown ion enhancement for the analyte. Further LLE using ether and different combinations of n-hexane and ethyl acetate (90–10 %, v/v), n-hexane and isopropyl alcohol (2–5 %, v/v) were tried but none of these was found suitable to give good and consistent recovery for all analytes. Finally, LLE using MtBE was tried and found suitable to give optimum recovery for LLM. The matrix effect was evaluated directly by extracting control drug-free plasma and then spiking with the analyte at the LLOQ concentration. There was no difference observed between the signal for the standard solution and the spiked extract at the LLOQ concentration. All frozen standards and samples were allowed to thaw at room temperature and homogenized by vortex mixing. Aliquots of 25 ll of spiked sample standards, QC samples, or unknown plasma samples were placed into a plastic 2-mL tarson tube, to which 0.5 mL of MtBE was added containing IS. This mixture was vortex-mixed for 5 min, and centrifuged for 10 min at 3,500 rpm. The organic layer was transferred to a glass tube and evaporated to dryness under a gentle stream of air at 40 °C. The dried residue was reconstituted in 100 ll of MP, vortexmixed for 30 s, and then transferred to micro-centrifuge tubes in which they were centrifuged for 5 min at 10,000 rpm. Finally, the supernatant was transferred to limited-volume injection vials, and 5 ll was injected into the LC–MS system.

Standards preparation

Validation

Standard stock solutions of LLM and IS were prepared in dimethyl sulfoxide (DMSO) to yield a concentration of 4 mM primary stock solution. These solutions were stored at 2–8 °C until use. The IS stock solution was diluted to achieve a final concentration of 25 lM with the diluent. Analytical standards for LLM were prepared in ACN: water (70:30, v/v) over a concentration range of 0.06–60.0 lM by serial dilution in blank rat plasma. QC samples at four different concentration levels (0.06, 0.15, 25.0, and 50.0 lM for LLM as LLOQ QC, LQC, MQC, and HQC, respectively) were prepared in three sets independent of the calibration standards (CS). Aliquots of prepared QC samples were stored at -70 °C until analysis. During analysis, these QC samples were spaced after every six to seven unknown samples.

The method was assessed in terms of specificity, accuracy, precision, recovery, sensitivity, linearity of detector response, and stability. LC–MS/MS analysis of the blank plasma samples showed no interference with the quantification of components LLM and the IS. On each validation day, CCs were analyzed simultaneously with QC samples. The procedure was performed on three different days using six replicates per standard concentration (0.06, 0.12, 0.23, 0.47, 0.94, 1.88, 3.75, 7.50, 15.00, 30.00, and 60.00 lM) and six replicates per QC concentration on each day.

(K2EDTA). Pooled plasma was prepared and stored at approximately -20 °C until needed. High-performance liquid chromatography and mass spectrometric condition

Sample preparation The next step was to develop simple and efficient sample clean up devoid of matrix effect and interference from endogenous plasma components for estimation of the

Specificity Ten blank samples from different six different rats were used for the chromatographic interference. Possible matrix effects were investigated by infusing a 0.06 lM solution of LLM (in MP) into the MS post-column, using a syringe pump via a tee. After a constant response was established, six different blank plasma samples were extracted and reconstituted, as detailed in ‘‘Experimental’’ section, and were injected into the LC–MS/MS system.

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Accuracy, precision, and recovery The intra-run and inter-run accuracy were determined by replicate analysis of the three QC levels along with the LLOQ level that were extracted from the sample batch. In each of the precision and accuracy batches, six replicates (n = 6) at each quality control inclusive of the LLOQ level were analysed. Accuracy is defined as the percent relative error (%RE) and was calculated using the formula % RE = (E-T) (100/T), where E is the experimentally determined concentration and T is the theoretical concentration. Assay precession was calculated using the formula % R.S.D. = (S.D/M) (100), where M is the mean of the experimentally determined concentrations and S.D. is the standard deviation of M. The extraction recovery of LLM from plasma was estimated at their respective low, medium, and high QC levels. Plasma samples (in six replicates) containing at QC concentration level were also spiked with IS at the working concentration of 25 lM. Samples were subsequently processed using the procedure described previously. A second set of plasma samples was processed and spiked postextraction with the same concentrations of the analyte and IS that actually existed in the pre-extraction spiked samples. Extraction recovery values for analyte and IS were determined by calculating the ratios of the raw peak areas of the pre-extraction spiked samples to that of the samples spiked after extraction.

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concentrations (% DEV). In order to establish the best standard curve for quantification (with or without IS), the simplest model tested with the lowest bias was used for further analysis. Stability Stability tests were performed to verify the stability of LLM during handling procedures. Samples were assayed at the two QC concentrations (0.15 and 50.00 lM). The samples were subjected to three freeze–thaw cycles with each freeze cycle lasting at least 12 h. The concentration of the drug after each storage period was related to the concentration of freshly prepared samples in the same analytical run. The stability of drug in the injection vials pending analysis was performed, where samples were prepared and placed in injection vials for 24 h before injection into the LC–MS/MS system. Analytes were considered stable if the concentration deviated less than ±20 % from the concentrations of freshly prepared samples. Pharmacokinetic analysis The suitability of the method for pharmacokinetic purposes was evaluated using plasma samples obtained from a male Sprague–dawley (SD) for up to 10 h after oral administration at 10, 50 and 100-mg/kg dose of LLM. A total of nine pharmacokinetic blood samples collected in 1.5-mL tarsons centrifuge tubes containing K2 EDTA as an anticoagulant. Blood samples were collected post dosing at 0.25, 0.50, 0.75, 1, 2, 4, 6, 8, and 10 h. Specimens were immediately centrifuged at 2,400 rpm for 10 min to separate the plasma, which was then stored at -70 °C until analysis.

Calibration curve Lower limit of quantification The LLOQ was defined as the lowest concentration of LLM that could be reliably and reproducibly measured with concentration determinations performed in replicates of at least six. To determine the LLOQ, pooled plasma samples were spiked to contain 0. 06 lM, and were run on four different days. The LLM peak was to be distinct from noise peaks and for verification of LLOQ, the peak area and peak height in a chromatogram from a pretreated LLM plasma sample containing 0.06 lM was compared with the noise signal. The LLOQ had to have a precision of B20 % and a signalto-noise ratio C3. Response function Calibration curves were constructed by least-squares linear regression analysis, where an eightpoint CC by plotting peak area ratio (y) of LLM to IS versus the LLM nominal concentration (x) without weighting, or using 1/x or 1/x2 as optional weighting factors. Calibrator response functions and choice of regression analysis were investigated by calculating correlation coefficients and the percent deviation for all standard

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Conclusion In the current report, an LC–MS/MS bioanalytical method development and validation of an analytical assay are described for the determination of LLM in rat plasma samples. The performance criteria for specificity, accuracy, precision, recovery, sensitivity, linearity, and stability have been assessed and accepted as being within the recommended guidelines of the FDA, indicating that the method can be used for determination of LLM in rat plasma. TLM derivative with similar structural nucleus to LLM was used as IS in assay method to account the variations due to matrix effect, extraction variability, and instrument performance with low sample volume and short run time.

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