METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATION OF TRIFLURIDINE IN HUMAN PLASMA BY USING LC-MS/MS TECHNIQUE

METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATION OF TRIFLURIDINE IN HUMAN PLASMA BY USING LC-MS/MS TECHNIQUE

Abdul Saleem Mohammad ^{* 1}, Fazil Ahmad ² and B. Jayanthi ³

Department of Pharmaceutical Analysis and Quality Assurance ¹, Department of Pharmacology ², Nizam Institute of Pharmacy, Deshmukhi - 508284, Telangana, India.

Department of Pharmacy ³, Annamalai University, Annamalai Nagar, Chidambaram – 608002, Tamil Nadu, India.

ABSTRACT: The main objective of the current study was to develop a highly sensitive LC-MS/MS (liquid chromatography-tandem mass spectrometry) technique for the quantification of trifluridine (TFD) in human plasma. Chromatography was processed on Phenomenex-RP-C18 (5 µm, 250 mm × 4.6 mm) column comprising acetonitrile, methanol and 5 mM ammonium formate (45:40:15, v/v) as a mobile phase, infused at a flow of 0.8 ml/min. TFD and internal standard ?-thymidine (TMD) were separated from 150 ml plasma utilizing Phenomenex cartridges. Quantification of the [M+H] +ion of analyte was done with MRM (multiple reaction monitoring) modes in LC-MS/MS utilizing electro-spray ionization. The parent and product ions transitions were monitored atm/z 297-181 and m/z² 41–42 for TFD and TMD respectively. The linearity was processed in the concentration range of 5.0–2000.0 ng/ml with regression coefficient (r²) = 0.999. Method intra and inter-batch precision findings were <3.33% and intra and inter-batch accuracy findings at three different concentrations were within 97.34% to 103.74%. Matrix effects were calculated by determining the %CV values for High and Low QC samples and was found to be 1.65% and 0.73% respectively. The % change for all the re-injected QC solutions was ≤7.45. The method was specific, accurate, precise and stable for more time and the technique was successfully applied for the quantification of trifluridine in human plasma samples.

Trifluridine, Antiviral, LC-ESI-MS/MS, Specificity, Accuracy and Matrix Effect

INTRODUCTION: Trifluridine chemically designated as 1- [4- Hydroxy- 5- (hydroxymethyl) oxolan- 2- yl] - 5- (trifluoromethyl)- (1H, 3H)-pyrimidine-2, 4-dione with molecular formula C₁₀H₁₁F₃N₂O₅ Fig. 1 and molecular weight 296.2 g/mol. This drug belongs to the nucleoside of fluorinated pyrimidine and structurally similar to idoxuridine.

In ophthalmic preparations, it acts against the virus to treat keratoconjunctivitis and also useful in epithelial keratitis caused by herpes simplex-virus type-I and type-II. This drug also useful in the treatment of metastatic colorectal cancer, when it combines with tipiracil (tablet) ^1-3. TFD acts by inhibiting viral replication.

The drug merges with DNA of the virus during the replication process and synthesis of an imperfect protein leads to an increase in the rate of mutations. TFD is also useful in cancer treatment through this mechanism only. The cancer cells will uptake and immediately phosphorylated by thymidine-kinase to active monophosphate. Subsequently, TFD-triphosphate was formed by phosphorylation and it directly merges into the DNA of cancer cells in thymidine place leads to inhibition of DNA functioning, DNA production, and proliferation of cancer cells. TFD mono-phosphate also prevents the thymidylate synthetase enzyme reversibly, which is useful in DNA replication ^4-6. Several RP-HPLC methods ^7-10 were reported in the literature of TFD for the quantification of TFD as single and with combined dosage forms. No single procedure was reported by utilizing LC-MS/MS technique. The goal of the present research was to develop a simple and highly sensitive bioanalytical LC-MS/MS technique for the quantification of TFD in human plasma samples.

FIG. 1: STRUCTURE OF TRIFLURIDINE

MATERIALS AND METHODS:

Reagents and Chemicals: Blank plasma samples in K3EDTA were acquired from Supratech Labs, Ahmedabad, India. Phenomenex (StrataXC-33 m) RP-extraction cartridges were acquired from Phenomenex, India. Trifluridine reference material of 99.62% and ?-thymidine internal standard (IS) of 99.89% purity were acquired from Biophore, Hyderabad, India. HPLC-grade methanol and acetonitrile and ammonium formate were procured from Sisco Laboratories, India.

Instruments: An LC-Shimadzu united with Shimadzu-LCMS triple quadrupole mass spectro-meter detection system (Shimadzu, Japan) was utilized. Chromatography was processed on Pheno-menex-RP-C₁₈ (5 µm, 250 mm × 4.6 mm) column comprising acetonitrile, methanol and 5 mM ammonium formate (45:40:15, v/v) as a mobile phase, infused at a flow of 0.8 ml/min. Analytical column-oven and auto-sampler temperatures were kept at 40 °C and 5 °C respectively. The electrospray ionization source was processed in positive mode of ionization.

Calibration Standards: TFD 1mg/ml stock solution was processed freshly by diluting 10mg drug in 10 ml of 70% methanol. Calibration standards of eight different concentration levels were processed by spiking blank plasma with TFD standard solution to get the concentrations of 5.0, 20.0, 100, 250, 500, 1000, 1500 and 2000 ng/ml.

Quality Control Standards: These solutions were processed at three different levels of Lowest Quality Control (LQC), standards, Median Quality Control (MQC) standards, and Highest Quality Control (HQC) standards. Quality control (QC) samples were processed according to calibration standards to get the concentrations of 20, 500 and 1800 ng/ml for LQC, MQC and HQC respectively. Processed solutions were kept in the freezer at-20 ºC till further analysis.

Mass Instrument Conditions: The mass instrument was operated in positive (+) ionization (PI) mode. The mass conditions were optimized based on tuning the instrument for TFD and IS by injecting the 500 ng/ml solution of both drugs at a flow of 10 µl/min into the LC mobile phase (0.20 ml/min). Optimized mass instrument settings for specific TFD and IS where: curtain gas (CG) was 21 psi, ISG (ion source gas) 1 was 40 psi, ISG (ion source gas) 2 was 40 psi, an ion-spray voltage was 4500 V, turbo-spray temperature at 550 ℃. Quantitative analysis was achieved by MRM of the transition pairs of transition sm/z 297–181 for TFD and m/z 241–42 for TMD, with 145 ms per transition as dwell time. TFD and IS precursor ions were formed by de-clustering potential (DP) s of 150 and 160 V respectively and precursor ions of TFD and IS were converted into fragments at collision energies (CE) of 25 and 25 EV with nitrogen (N₂) gas at a pressure of 5 arbitrary units.

Protocol for Sample Separation: 25 ml of TMDIS working standard solution was mixed with 150 ml of spiked plasma samples and sonicated for 2 min. 500 ml of water was mixes to the resultant solution and subjected to vortex. The resulting solution was filled in Strata XC-33 m separation cartridge, which was conditioned by utilizing 1 ml of methanol and subsequently by 1 ml water. Sample washing was processed by 1.0 ml of water, subsequently drying of the cartridge by N₂ gas (1.72 × 10⁵ Pa) for 2.0 min.

Separation of TFD and TMD was processed by utilizing 500 ml of mobile phase into pre-labeled vials. From the resultant solution, 10.0 ml was utilized for infusing into the LC-system.

Method Validation: Complete TFD-method validation in human K3EDTA-plasma was processed, as per USFDA and EMEA guidelines. The technique was validated for specificity, matrix effect, precision, linearity, sensitivity, process efficiency, accuracy, reinjection reproducibility and dilution integrity and stability study of TFD ^11-14.

Selectivity and Sensitivity: Analytical technique selectivity concerning matrix metabolites, constituents, and associated medicaments were evaluated after analyzing 10 lots (lipemic-2, haemolyzed-2 and normal-6) of human K3EDTA-plasma. The resulting processed samples were extracted with SPE and analyzed for TFD at LOQ level. The peak response of all the matrix constituents in the blank sample at TFD and IS retention times should be <20 and 5% of the average peak response of TFD and IS at LOQ level respectively. The sensitivity was estimated by assessing signal to noise-(S/N) ratio in 10 batches of screened and spiked LOQ-samples. The S/N was measured by the formula:

S/N ratio = (Signal: noise ratio of LOQ) / (average signal: noise ratio of blanks) > 5

Dilution Integrity: It was processed by making the sample concentration nearly two times the 90% ULOQ concentration. The resultant solution was made dilution (2 and 4 times) with blank plasma to get the solution concentration of solution within linearity range.

Then the samples were evaluated against a fresh calibration standard solution. The acceptable norms were the same as precision and accuracy parameter.

Linearity: Calibration standards (Non-zero) of eight different concentrations of 5.0, 20.0, 100, 250, 500, 1000, 1500 and 2000 ng/ml were freshly processed and quantified in 3 individual runs.

Linear plots (Peak area ratio of analyte and IS peaks versus nominal concentration) were plotted by the least-squares linear regression and reciprocal of the squared concentration (1/x²) used as a weighting factor.

Method Precision and Accuracy: The inter-day and intraday precision and accuracy were processed for TFD in human plasma. Within a day intra-run and between days, inter-run accuracy was analyzed for six replica samples of Quality control at LLOQ, Low, median and high QCs. Method precision was evaluated by determining the % Coefficient of variation for all control samples. The percentage deviation should be <15.0. In the same way, the average accuracy should be ± 15 ^15-18.

Matrix Effect: Each of LQC and HQC samples after extraction in 6 different blank matrix lots (post-extraction spiked samples) were prepared. Simultaneously, SIX replicates of equivalent aqueous/neat QC samples were prepared and analyzed ^19-21. Matrix factor for the analyte and IS in each lot were evaluated using the formula:

Matrix Factor = Peak response in the presence of matrix ions / Average peak response in aqueous samples

Reinjection Reproducibility: This parameter was processed by re-injecting QC-samples from accepted precision and accuracy lot during validation. The re-injected sample concentrations were evaluated by comparing with calibration standard solutions of the same accuracy-precision lot which were estimated 48 h before. The percentage difference between re-injected and original values was measured by the formula:

% difference = Actual concentration-reinjected concentration / (Actual concentration)   × 100

Stability: Each six low and high QCs were regained from the freezer after 3 freeze-thaw phases. Samples were kept at -30 °C in three cycles of 24.0, 48.0, 72.0 h. Bench-top stability was assessed for a 6.5 h period with standard concentrations. For the long-term stability quality control standards were determined by analysis after 121 days kept at -50 °C. Stability solutions were prepared and separated along with freshly spiked CSs. The accuracy and precision of the stability solutions should be ± 15% of their nominal concentrations. The auto-sampler stability esti-mated after 72 h under auto-sampler (at 10 °C) condition. The freeze-thaw stability was performed by storing the QC solutions at -50 °C (frozen) and thawed at room conditions for 3 times. The change in analyte concentration was less (<15%) then the compound said to be stable ¹⁷.

Ruggedness: Method ruggedness was assessed by processing QC standards for one precision and accuracy batch utilizing different columns of same composition by different analysts. The % RSDs for LQC, MQC and HQCs should be ≤ 20% for LLOQ and ≤ 15% for the remaining QC standards.

RESULTS AND DISCUSSION:

Internal Standard Selection: Identification and selection of IS was a very important thing in an LC-MS/MS technique. The IS should have similar mass and chromatographic behaviour with an analyte to be determined. Therefore, TMD chosen for TFD internal standard ^{18, 20}.

Optimization of Chromatography: To get a good resolution between TFD and TMD different phenyl and C18-columns like Ascentis, Zorbax SB-C18, Hypurity C-18, Poroshell-EC-C18, Sunshell-C18, Kinetex-C18, Discovery C18, Luna-RP-C18 and ACE-RP-C18 were utilized. TFD and IS were well separated using Phenomenex-RP-C18 (5 µm, 250 mm × 4.6 mm) column. However, a mobile phase comprising acetonitrile, methanol and 5 mM ammonium formate (45:40:15, v/v) was established optimal.

Method Validation: Method was validated as per the EMEA and USFDA-guidelines and around was no nosiness noticed at the RT (retaining time) of TFD and TMD in the lots of plasma.

The blank, LLOQ, Low, Medium and High QC chromate-grams were represented in Fig. 2 and 3. The S/N-ratio during the method validation was found to be more than 25, which was acceptable in accordance with the USFDA and EMEA-guidelines.

FIG. 2: REPRESENTATIVE CHROMATOGRAMS OF BLANK (A) AND SPIKED LLOQ-SAMPLE (B) OF TRIFLURIDINE

The drug has a limit of quantitation value of 5.0 ng/ml and the precision and accuracy values were found to be 8.62% and 98.12% at LLOQ concentration level. The linearity graph was linear over the concentration range of 5.0–2000.0 ng/ml for TFD. Linearity graph was made using the peak response ratio of drug to IS and the ‘R²’ value was estimated and the value was more than 0.99 and findings were shown in Table 1. Accuracy and precision were processed, and the findings were tabulated in Table 2. The inter-day and intra-day precision were measured in % CV and the values were found between 0.42% to 3.33% and the inter-day and intra-day accuracy findings were present between 97.34% to 103.74%. Matrix effect was estimated by determining the % CV values for High and Low QC samples and was found to be 1.65% and 0.73% respectively. The findings were shown in Table 3.

Stability studies of TFD were processed for Auto-sampler stability (10 °C, 76.90 h), Benchtop stability (ice-cold water, 6.5 h), Freeze and thaw stability (three freeze-thaw cycles), Long-term stability (−50 °C, 121 days) and all the findings were shown in Table 4. The detected average nominal concentrations of TFD were within ± 15% of their particular nominal concentration. There was no change in the concentration of TFD drugs in human K3EDTA for 2.0 h. Method ruggedness for TFD was processed and estimated. The % RSD findings were calculated for the same and were represented in Table 5.

Method Re-injection reproducibility was proven by re-injecting quality control solutions of precision and accuracy lot three and quantified against the actual estimated linear graph of precision and accuracy lot three. The % change for all the re-injected QC solutions was ≤7.45.

FIG. 3: CHROMATOGRAMS OF TRIFLURIDINE SPIKED (A) LQC, (B) MQC AND (C) HQC SAMPLES

TABLE 1: LINEARITY OF TRIFLURIDINE

CV- coefficient of variance; SD- standard deviation

TABLE 2: TRIFLURIDINE INTRA AND INTER-DAY QUALITY CONTROL STANDARDS

SD- standard deviation; CV- coefficient of variance

TABLE 3: MATRIX EFFECT RESULTS FOR TFD

SD- standard deviation; CV- coefficient of variance

TABLE 4:  TRIFLURIDINE STABILITY DATA

CV: Coefficient of variation

TABLE 5: TRIFLURIDINE RUGGEDNESS DATA

n- 6 replicates; RSD- Relative standard deviation

CONCLUSION: A simple and sensitive bioanalytical LC-MS/MS technique for the quantification of TFD in human plasma was developed and validated as per the USFDA and EMEA guidelines. The drug has a limit of quantitation value of 5.0 ng/ml and the precision and accuracy values were found to be 8.62% and 98.12% at LLOQ concentration level. The % change for all the re-injected QC solutions was ≤7.45.  Matrix effect was estimated by determining the % CV values for High and Low QC samples and was found to be 1.65% and 0.73% respectively. The inter-day and intra-day precision were measured in % CV and the values were found between 0.42% to 3.33% and the inter-day and intra-day accuracy findings were present between 97.34% to 103.74%. The drug has more stability under different stability conditions. This developed analytical technique was useful in the routine quality control of TFD in human plasma samples.

AUTHORS’ CONTRIBUTIONS: All authors contributed equally to this manuscript.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTERESTS: There are no conflicts of interest.

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    How to cite this article:

    AS Mohammad, Ahmad F and Jayanthi B: Method development and validation for the quantitation of trifluridine in human plasma by using LC-MS/MS technique. Int J Pharm Sci & Res 2020; 11(7): 3252-59. doi: 10.13040/IJPSR.0975-8232.11(7).3252-59.

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