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Methotrexate is an antimetabolite and antifolate drug. 66 It is used in treatment of cancer, autoimmune diseases, ectopic pregnancy, and for the induction of medical abortions. It acts by inhibiting the metabolism of folic acid. Folic acid is needed for the de novo synthesis of the nucleoside thymidine, required for DNA synthesis. Also, folate is needed for purine base synthesis, so all purine synthesis will be inhibited. MTX therefore, inhibits the synthesis of DNA, RNA, thymidylates, and proteins67 68.
Philadelphia chromosome/BCR-ABL-positive acute lymphoblastic leukemia (ALL) is the largest genetically defined subtype in adult ALL. Several strategies have been tested to optimize the combination of DSN and chemotherapy 60, 69-70. The combination of dasatinib with a variety of cytotoxic chemotherapy regimens both in younger and elderly patients with de novo or minimally pretreated Philadelphia positive ALL was explored in recent clinical trials. DSN and MTX combination have advanced to phase II and phase III clinical program particularly in Hyper-CVAD and Dasatinib in Patients with Philadelphia Chromosome Positive and/or BCR-ABL Positive ALL 71.
During literature survey, we found several publications quoting the methods for the determination of either individual or simultaneous estimation of multiple analytes in biological fluids by reverse phase chromatography HPLC -UV and LC-MS/MS 62, 72-80. However, these methods showed limited extraction procedure, and required a relatively long analysis time to attain sufficient chromatographic separation. To our knowledge, no prior reports have described a LC-MS/MS-based method for simultaneous determination of MTX, DSN and its active metabolite M-4 from plasma. Hence we developed a reverse phase HPLC method for simultaneous estimation of MTX, DSN and M-4 on C18 column using tandem mass spectroscopy detection and validated before using in our preclinical experiments. The current study describes a fast, sensitive, specific and simple liquid-liquid extraction method using LC-MS/MS for the simultaneous determination of MTX and DSN along with its major active metabolite M-4 in rat plasma suitable for pharmacokinetic and drug-drug interaction studies. The method was validated using authentic pure standards. This method was successfully applied in the pharmacokinetic drug-drug interaction study of MTX with DSN in rats after oral administration in combination.
5.2. Materials and Methods
5.2.1 Reagents and standards
Pure reference standards of methotrexate (MTX) and tolbutamide (IS) were obtained from Sigma-Aldrich (Germany). Dasatinib (DSN), N- dsehydroxyethyl dasatinib (M-4) was obtained from Incozen Therapeutics Pvt Ltd. Hyderabad. Acetonitrile (HPLC grade), methyl t-butyl ether (TBME), methanol (HPLC grade) and ammonium acetate (GR-grade) was procured from E Merck (India) Ltd., India. Formic acid was obtained from Sigma Aldrich, Germany. Ultra pure water of 18 MÎ©/cm was obtained from Milli-Q purification system (Millipore, USA). Blank rat plasma was collected from healthy, drug free Wistar rats at the Incozen Therapeutics Private Limited (India). Plasma was obtained by centrifuging the K2-EDTA (di-potassium ethylene diamine tetra acetic acid, Sigma Aldrich, Germany) blood at 3000 rpm for 10 min.
For pharmacokinetic studies, young and healthy male wistar rats weighing 200 ± 30 g were obtained from Mahaveera Enterprises, Hyderabad and housed at Incozen Therapeutics Pvt Ltd, Hyderabad in appropriate stainless steel cages in standard laboratory conditions with regular 12 h day-night cycle in well-ventilated room with an average temperature of 24-27°C and relative humidity of 40-60%. Standard polluted laboratory chow (Providing Animal Nutrition India Private Limited, Bengaluru, India) and water allowed ad libitum to rats. All applicable ethical approval, guidelines for maintenance and experimental studies with wistar rats were followed.
5.2.3. Stock solution, calibration standards and quality control samples
Standard stock solutions of MTX, DSN, M-4 and IS were prepared in methanol with a final concentration of 1 mg/ml. These solutions were stored at 2-8°C until use. The IS stock solution was diluted to achieve a final concentration of 250 ng/mL with the diluent (acetonitrile: water, 70:30 v/v) solution. Analytical standards for MTX, DSN and M-4 were prepared in acetonitrile: water (70:30, v/v) over a concentration range of 0.97 ng/mL to 931.37 ng/mL, 1.04 ng/mL to 1000.05 ng/mL and 0.95 ng/mL to 910.56 ng/mL respectively by serial dilution, and same concentration range for calibration curve were prepared in blank normal rat plasma. Quality control (QC) samples at four different concentration levels (0.98, 2.81, 374.82, 749.64 ng/mL for MTX, 1.05, 3.02, 492.86, 805.72 ng/mL for DSN and 0.96, 2.75, 366.44, 732.89 ng/mL for M-4 as lloqc, low , medium and high, respectively) were prepared in three sets independent of the calibration standards. During analysis, these QC samples were spaced after every six to seven unknown samples.
5.2.4. Sample preparation
An aliquot of 50 µl was plasma was transferred to a 2 mL eppendrof micro centrifuge tube, 100 µl of IS and 1mL of TBME were added and sample was vortex mixer for 3 mins. After centrifugation of the sample at 14000 rpm for 5mins, supernatant was collected in to 3 mL test tube. Then it was dried to residue under nitrogen stream. (Nitrogen Evaporator, Caliper Instruments, USA). The residue was reconstituted in 100 µl of mobile phase and 5µl was injected onto the LC-MS/MS system.
5.2.5. Chromatographic condition
A Shimadzu SIL - 20 AC HT (Shimadzu Corporation, Japan) consisting of flow control valve, vacuum degasser operated in isocratic mode to deliver the mobile phase at flow rate of 1 mL/min. The chromatographic system consisted of reverse phase C18 column (50mmÃ- 3mm i.d., 4.6µ) (YMC-PACK, Japan) and mobile phase consists of methanol (A), 2 mM ammonium acetate buffer (B), (pH ~3.2 adjusted with 0.1% formic acid). Gradient programs were set as from 0.0 to 0.8 min 90% of A: 10% of B, from 0.8 to 3.2 10% of A: 90% of B and 3.2 to 5.0 min 90% of A: 10% of B was maintained to run the chromatography. The samples (5 µL) were injected on to the LC-MS/MS system through an auto injector. The auto sampler temperature was kept at 10°C and the column oven was maintained at 40°C.
5.2.6. Mass spectrometric condition
Mass spectrometric detection was performed on Thermo Scientific - Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped with a Heated Electron Spray Ionization (HESI) source (San Jose, CA, USA), a Selected Reaction Monitoring (SRM) mode was used for data acquisition with Xcalibur 1.2 software(Thermo- Scientific, San Jose, CA, USA). Peak integration and calibration were carried out by using LC Quan 2.5.2 software (Thermo- Scientific). MS and MS/MS condition for pure standards of MTX, DSN, M-4 and IS were optimized by continuous infusion at 5µl/min using inbuilt syringe pump. The transitions monitored were m/z 455.0>175.0, 488.1 >401.0, 444.26>401.0, and 271.1>-155.0 for components MTX, DSN, M-4 and IS, respectively. All analyses were carried out in positive ion HESI with spray voltage set at 4500 V. The heated capillary temperature was set 350°C. Nitrogen sheath gas and auxiliary gas were set at 40, 30 kPa. The argon gas collision induced dissociation was used with a pressure of 1.6m Torr and the energy selected to be 3500 eV. Total run for an LC-MS/MS analysis was 5.0 min.
5.2.7. Application to pharmacokinetic study
The method was successfully applied to generate the plasma concentration versus time profile of test drugs (MTX and DSN) as well as to detect its metabolite (M-4) in plasma following simultaneous intra-peritoneal administration at 4 mg/kg dose of MTX and 10 mg/kg oral administration dose of DSN in male wistar rats. Intra-peritoneal formulation prepared using water for injection to achieve 4 mg/ kg body weight of animal. Oral formulations were prepared in suspension form by triturating accurately weighed amount of powdered compound in methyl cellulose solution (0.5%, w/v water) in gravimetric dilution pattern. Oral dose of DSN 10mg/kg body weight of animal was administered using an oral gavage at 5 ml/kg volume in rats after overnight fasting (12 hr) and these animals were continued for fasting till 4 hr post dose. The blood samples (0.15 ml) were collected from retro orbital sinus at predose, 10, 15, 30 min and 1, 2, 3, 4, 6, 8, 10 and 24 hrs post dose and were kept on ice bath till further processing. These samples were separated for plasma by centrifuging at 4°C for 10 min at 3000 rpm and then stored at -70°C till further analysis. These samples were analyzed for simultaneous estimation of the levels of MTX, DSN and its active metabolite M-4.
Pharmacokinetic parameters, including the area under the concentration-time curve (AUC), maximum plasma concentration (Cmax) and time to reach the maximum
concentration (Tmax), were estimated by means of a non-compartmental analysis using
Phoenix WinNonlin (Pharsight Inc., USA, version 6.1).Statistical parameters like mean,
Standard deviation and C.V were calculated by using MS-Excel 2007 (Microsoft®). Incurred sample reanalysis (ISR) was performed to reconfirm the initial values and to demonstrate that the assay was reproducible. In the study, ISR was performed on 18 plasma samples from six different rats at Tmax and the second time point covering the phase of elimination.
5.3. Results and discussion
5.3.1. Mass spectrometry
In order to find most sensitive ionization mode for the components studied, ESI positive ion mode and ESI negative ion mode were tested with various combination of mobile phase, i.e. , methanol, acetonitrile and water/ammonium acetate buffer (2 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 ion mode were 3-10-fold higher for all components in analyses using methanol: ammonium acetate buffer (2 mM), versus experiments run with ESI negative ion mode. The protonated molecular ion of [M + H] +, m/z 455.0, 488.1, 444.2 and 271.1 amu were obtained for MTX, DSN, M-4 and IS, respectively. No significant solvent adduct ions or fragment ions were observed in the full scan spectra of all the compounds. 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. Representative spectra and fragment pattern showed in the Figure.1. The optimized tubelens potentials for the protonated [M+H] + of component MTX, DSN, M-4 and I.S., were found to be 124, 132, 176 and 105 eV respectively.
5.3.2. Liquid chromatography
Methanol rather than Acetonitrile was chosen as an organic modifier because of its better peak shape. Moderately high acidic 2 mM ammonium acetate buffer pH~3.2 was required to achieve acceptable peak width and shapes. A reverse phase C18 column (50mmÃ- 3mm i.d., 4.6µ), YMC-PACK, Japan with methanol: ammonium acetate buffer in gradient mode was applied in final LC method. Within the total analysis time of 5 min, all components were eluted in 0.6 -2.8 min.
5.3.3. Optimization of LC-MS/MS condition
Final SRM transitions were selected on the basis of signal to noise ratio (S/N) ratio with on-column injection analysis. Nitrogen sheath gas, auxiliary gas, argon gas collision induced dissociation, ion spray voltage and temperature were set to 40, 30 KPa, 1.6m Torr and the energy selected to be 3500 eV. and 350°C, respectively. The transitions selected were m/z 455.0>175.0, 488.1 >401.0, 444.26>401.0, and 271.1>-155. For components MTX, DSN, M-4 and IS respectively. The fragment pattern ions selected for final SRM method are given in Fig. 1.
5.3.4. Sample clean up
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 analytes in rat plasma. Hence the precipitation method was tried initially with acetonitrile (1.5 mL) and it has shown ion enhancement for all the analytes. Further liquid-liquid extraction (LLE) using ether and different combinations of hexane and ethyl acetate (80-20%, v/v), n-hexane and isopropyl alcohol (2-5%, v/v) was tried but none of these was found suitable to give good and consistent recovery for all analytes. Finally, LLE using TBME was tried and found suitable to give optimum recovery for all analytes. For determination of matrix effect, control drug free plasma was extracted using TBME as described above and final supernatant was evaporated to dryness. Dry extracts were dissolved using analytes and IS standard solutions at LOQ concentration level that represent 100% recovery. Matrix effect was determined by comparing the analytical response of these samples with that of standard solutions.
5.4. Method validation
Accuracy, precision, selectivity, sensitivity, linearity and stability were measured and used as the parameter to assess the assay performance. LC-MS/MS analysis of the blank plasma samples showed no interference with the quantification of components MTX, DSN, M-4 and the IS. The specificity of the method was established with pooled and individual plasma samples from six different sources. The retention times of all the analytes and the IS showed less variability with a percent co-efficient of variance (%CV) well within acceptable limits of 5%.
5.4.1. Limit of detection (LOD) and quantification (LOQ)
Two criteria were used to define LOQ, i.e., (1) the analytical response at LOQ must be five times the baseline noise and (2) the analytical response at LOQ can be detected with sufficient accuracy (80-120%) and precision (20%). LOD is defined as the lowest concentration of the analyte at which the signal is larger than three times the baseline noise. The measured LOQ and LOD values were 20 and 5 arbitrary units for all three analytes. The limit of quantification (LOQ) was set at 1.0 ng/mL. These results well met the requirements of quantifications of all analytes in plasma
The peak area ratios of analytes to IS in rat plasma were linear over the concentration range 0.97 ng/mL to 931.37 ng/mL for MTX, 1.04 ng/mL to 1000.05 ng/mL for DSN and 0.95 ng/mL to 910.56 ng/mL for M-4. 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 best fit for the calibration curve could be achieved by a linear equation of y = mx + c and a 1/x2 weighting factor for all components. The correlation coefficients (R2) for all components were above 0.996 over the concentration range used.
Figure . Total Ion Chromatogram and fragmentation pattern of MTX, DSN, M-4 and IS
Table . Summary of precision and accuracy from QC samples in wistar rat plasma
Measured concentration (ng/ml)
(mean ± SD)
Measured concentration (ng/ml)
(mean ± SD)
5.4.3. Precision and accuracy
The intra-day precision (expressed by coefficient of variation of replicate analyses) was estimated on the four quality control levels and the within batch precision on the ten calibration standard levels. Table 1 shows the results obtained for the within batch and between batch precision for MTX, DSN and M-4. The precision for all these analytes under investigation were not exceeded 15% at any of the concentrations studied and well met the requirements of validation
The recovery of MTX, DSN and M-4 from plasma was estimated at their respective low, medium and high QC levels. Plasma samples (in six replicates) containing all analytes at QC concentration level was also spiked with respective internal standards. The absolute recoveries ranged from 79.4 to 87.2, 88.2 to 93.1% and 94.1 to 85.2% for of MTX, DSN and M-4 respectively. The results are indicated in Table 2.
Table . Extraction recovery in rat plasma (n=6)
QC samples were subjected to short term and long term storage condition (âˆ’70°C), freeze-thaw stability, auto-sampler stability and bench-top stability studies. 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 (25°C) for 6 hours. Freeze-thaw stability of low and high QC samples was evaluated after 3 freeze thaw cycles. The autosampler stability was studied for low and high QC samples stored at autosampler at 10°C for 24 hour. 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 results indicated that each analyte had an acceptable stability under those conditions, as shown in Table 3.
5.4.6. Sample dilution
To demonstrate the ability to dilute and analyze samples containing all analytes at concentration above the assay upper limit of quantification, a set of plasma samples was prepared containing MTX, DSN & M-4 at a concentration of 2794.1, 3003.1 & 2731.7 ng/mL respectively, and placed in a -70°C freezer overnight prior to analysis. After thawing, certain aliquot was diluted either with 4 & 8 times wistar rat plasma and analyzed respectively. 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.
Table . Stability in rat plasma (n=6)
Bench top stability a
Freeze-thaw stability c
30 days storage stability d
a Exposed at ambient temperature (25°C) for 6h; b Kept at autosampler temperature (10°C) for 24h; c After three freeze-thaw cycles; d Stored at -70°C
5.4.7. Comparison of methods
Most of the available methods for determination of MTX were involved using gas chromatography and LC-MS/MS 72-73, 75, 81. Previous methods describing for the determination of DSN and its metabolites involved solid phase extraction (SPE) using LC-MS/MS 80 Other methods using HPLC-UV and LC-MS/MS were involved with various gradient and isocratic conditions comprised of multiple steps of LLE or SPE procedures for estimating DSN 76, 79. However, the reported method involves complex step extraction procedures and larger run times. The aim of present investigation is to develop and validate a simple LC-MS/MS method using isocratic mode with sufficient accuracy and precision for simultaneous estimation of MTX & DSN along with its active metabolite M-4 and its subsequent use in pharmacokinetic studies in rats. The present method involves simple LLE procedure with good sensitivity and an isocratic reverse-phase HPLC analysis for all analytes of interest. This method is specific for MTX & DSN and its major active metabolite M-4 with no interference and with good linearity, accuracy and precision. This method involves only 50µl blood plasma and in our LLE extraction procedure we achieved a high level of extraction efficiency for the analyte(s). This makes the assay highly reproducible and allows us to lower the limit of quantification. Furthermore, this one-step LLE extraction procedure uses less single solvent which decreases both the cost and duration of the assay. The chromatographic conditions of this method were optimized for a 5 min run time on LC-MS/MS.
5.5. Application to pharmacokinetic study
The method described above was successfully applied to a PK drug-drug interaction study in which plasma concentration of pure markers was determined for up to 24 h after Simultaneous intra-peritoneal administration at 4 mg/kg dose of MTX and 10 mg/kg oral administration dose of DSN in male wistar rats. The plasma concentration time profiles of MTX, DSN & its active metabolite M-4 are shown in Figure 2, and could be traceable up to 24 h, 12h and 12h respectively. The pharmacokinetic parameters of MTX, DSN and M-4 are presented in Table 4. During ISR it was observed that all of the samples were within ±20% of initial concentration value, further demonstrating that this method is capable of producing reproducible results over time.
Table 4. Pharmacokinetic parameters after single dose oral administration of MTX and DSN simultaneously in wistar rats
Figure . Mean plasma concentration vs. time after single dose oral administration of MTX and DSN in wistar rats (n=6)
An LC-MS/MS bioanalytical method for simultaneous determination of three analytes, MTX, DSN and M-4 was developed and validated in rat plasma. The method was good enough to detect low concentration of 1 ng/mL for all these analytes in 50 µL rat plasma and further can be improved by increasing the plasma volume. Analytes recovery from spiked control samples were >79%, convenient and fast LLE method. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% at low and <10% at other concentrations. The method was successfully applied to generate stability profile as well as PK evaluation of simultaneous administration of MTX, DSN in rat following oral administration.