DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATION OF TENOFOVIR AND EMTRICITABINE

Objective: Therapeutic drug monitoring of tenofovir and emtricitabine, two commonly used antiretroviral drugs, is important to maximize effectiveness while minimizing side effects. Materials: A Liquid Chromatography-Mass spectrometry (LC-MS/MS) method was developed to quantify tenofovir and emtricitabine in human plasma samples. The method involves a simple solid phase extraction procedure followed by liquid chromatography separation using a Penta Fluoro Phenyl (PFP) column with a Phenomenex C18 column and a mobile phase of ammonium formate, acetonitrile, and methanol, achieving separation in under 4 min. Results: The method showed good accuracy, low limits of quantification, adequate recovery, minimal matrix effects, and specificity. Analyte stability under multiple storage conditions was demonstrated. Conclusion: The validated LC-MS/MS method provides a reliable tool for therapeutic drug monitoring and pharmacokinetic studies of anti-Human Immuno-deficiency Virus (HIV) regimens. The assay can be applied to large populations, especially in resource-poor settings, to help individualize dosing and improve clinical outcomes while reducing toxicity.


INTRODUCTION
Liquid Chromatography-Mass spectrometry (LC-MS) coupled with a Turbo spray interface (TSI) provides a sensitive and powerful technique [1,2].LC separates mixtures into components, while MS identifies components with high specificity and sensitivity.The coupled technique can analyze biochemical, organic, and inorganic compounds in complex samples [3,4].
Tenofovir and emtricitabine inhibit Human Immuno-deficiency Virus (HIV) reverse transcriptase and lower the amount of HIV in the body.They are administered together as part of highly active antiretroviral therapy (HAART) [5,6].A bioanalytical method is needed to determine drug concentrations in biological fluids like plasma for purposes of therapeutic drug monitoring and pharmacokinetic studies.LC-MS/MS method provides a significantly faster run time compared to other bioanalytical methods, allowing high-throughput analysis for therapeutic drug monitoring and pharmacokinetic studies.Moreover, the LC-MS/MS technique offers high sensitivity and specificity advantageous for analysis in biological matrices.The proposed method once validated, provides an efficient tool to optimize anti-retroviral therapy through individualized dosing for large patient populations, especially in resource-limited settings [7,8].Method development and validation ensure a reliable technique under given laboratory conditions using available resources.This includes demonstrating that the technique is reproducible and fit for its intended use in analyzing drug levels in biological matrices [9,10].The main objective of this study is to develop and validate an efficient LC-MS/MS method for the simultaneous quantification of tenofovir and emtricitabine in human plasma samples to optimize antiretroviral therapy.

Materials
The analytes, tenofovir and emtricitabine, and their internal standards (tenofovir-d7 and emtricitabine-d2) were procured from Clear synth Ltd, Hyderabad.Methanol and acetonitrile are obtained from Thermo Fischer Scientific, Hyderabad, while ammonium formate, formic acid, and ammonia solution are obtained from Merck Specialities Ltd, Kakinada.

Blank plasma
K2 Ethylene Diamine Tetra-acetic acid (EDTA) human blank plasma was sourced from Doctor's Pathological Lab in Hyderabad.This plasma was used for method development, validation, and analysis.This technique was utilized to generate calibration standards and quality control samples.The plasma was collected from healthy, non-smoking volunteers who provided informed consent for use for research purposes.Blood samples were collected in K2 EDTA vacutainers from the volunteers [11].

Instrumentation
The study utilized the following instruments for the analysis.An analytical balance model, CP225D from Sartorius, was used.A centrifuge model SW12R from Firlabo, France, was employed.Deep freezers at-86 °C and-20 °C of the VIP series from Sanyo, USA, were used to store samples.A High-Performance Liquid Chromatography (HPLC) system from Shimadzu, Japan, comprising LC-20 AD pumps, DGU-20 A3 degasser, CTO-ASVP column oven, and SIL HTC autosampler was utilized.A microbalance model CP2P from Sartorius, Germany, was used.Micropipettes of 5-50 μl and 100-1000 μL from Brand, Germany, were employed.An API-4000 mass spectrometer with Turbo ion spray interface in negative ionization mode from MDA, Sciex, Canada, was used.A Turbo Vap LV nitrogen evaporator from Caliper Life Sciences, USA, was used.An Orion star pH meter from Thermo Electron Corporation, USA, was employed.A SpeeDisk-48 solid phase extraction unit from Orochem Technologies, USA, was used.A Powersonic 510 ultrasonic bath from Hwashin Technologies, Korea, and a Spinix vortex from Tarsons, India, were utilized.Purified water was obtained from water purification units, including the Elix 10 and Milli-Q gradient A10 from Millipore, USA.Data acquisition and processing on the LC-MS/MS instrument were performed using Analyst software version 1.5.1.

Chromatographic conditions
The chromatographic conditions were optimized through a trial and error or scouting approach.Various parameters were varied and tested to determine the optimal conditions for best performance [12,13].The mobile phase consisted of 5 mmol ammonium formateacetonitrile-methanol 60:25:15 (v/v/v).A Kinetex PFP, 50 X 4.6 mm, 5μm (Phenomenex) column was used.The flow rate was 1.0 ml/min with an injection volume of 10 μl.The column oven temperature was 35±1 °C, and the autosampler temperature was 5±1 °C.The retention times were tenofovir: 1.53 min., emtricitabine: 2.14 min., tenofovir-d7: 1.54 min internal standard (ISTD), and emtricitabine-d2: 2.14 min (ISTD) and the run time was 3.2 min with a splitness of 50:50.

Mass spectrometric conditions
For mass spectrometric detection, a turbo ion spray interface operated in positive ion mode was used [14,15] The dwell time was set to 300 ms for each transition.Quadrupoles 1 and 3 were operated at unit resolution.The gas/turbo ion spray source parameters were optimized as follows.Nitrogen was used as the ion source gas 1 and was set at 35 psi to aid in analyte spray formation [16].Nitrogen was also used as ion source gas 2 at 45 psi to stabilize the ion spray.A curtain gas flow of 30 psi of nitrogen was used to improve ion transmission into the mass spectrometer.The collision gas was set at 10 psi of nitrogen for collision-induced dissociation.The ion spray voltage was set to 5000 V to produce maximally charged droplets.The source temperature was maintained at 500 °C to assist desolvation of ions and droplets.The interface heater setting was turned on to further aid the desolvation of ions entering the mass spectrometer [17].The mass spectrometric parameters were optimized to achieve maximum sensitivity.The declustering potential was 45V for tenofovir and tenofovir-d7 while it was 52V for emtricitabine and emtricitabine-d2.An entrance potential of 10V was applied for all analytes.A collision energy of 29V was used for tenofovir and tenofovir-d7 while 32V was used for emtricitabine and emtricitabine-d2.The collision cell exit potential was 13V for tenofovir and tenofovir-d7 and 20V for emtricitabine and emtricitabine-d2.

Stock solutions and calibration standards
Individual stock solutions of 1 mg/ml tenofovir and 1 mg/ml emtricitabine were prepared in methanol to quantify the drugs in human plasma using LC-MS/MS [18,19].Serial dilutions of these stock solutions were performed to obtain calibration standards and quality control samples.Separately, internal standard stock solutions of 2 mg/ml tenofovir-d7 and 2 mg/ml emtricitabine-d2 were diluted to achieve a 500 μg/ml working concentration of each internal standard [20].The stock solutions were stored at 2-8 °C and found to be stable for 22 d [21].
Calibration curves ranging from 5 to 500 ng/ml for tenofovir and 8 to 4000 ng/ml for emtricitabine were made.Quality control plasma samples were also prepared to standardize the method developed.
Desired drug levels in plasma were achieved by spiking with 1% of the working standard dilutions.Long-term stability quality control samples were prepared in pooled plasma and stored at-70 °C.For sample preparation, 400 μL of plasma was mixed with 100 μL of the 500 μg/ml internal standard solution.Then 0.4 ml of 0.2% formic acid was added and loaded onto preconditioned Phenomenex Strata-X solid phase extraction cartridges.The cartridges were washed and eluted with 1 ml of 2% ammoniated methanol.The eluate was dried under nitrogen, reconstituted in 400 μL mobile phase, and 10 μL injected into the LC-MS/MS system [22].

System suitability and repeatability assessment
To assess that the LC-MS/MS system and method were functioning properly, 2 injections of a low-concentration calibration standard and 6 injections of a high-concentration calibration standard were performed for tenofovir and emtricitabine [23].The low concentration standard was injected to inspect the peak shapes.The coefficient of variation (%CV) of the area ratios from the six highconcentration standard injections should be less than 4% according to guidelines [21].

Carryover and cleaning validation
To evaluate the autosampler performance in terms of carryover, extracted blank samples, high-standard samples, and low-standard samples were injected.The carryover should not be more than 20% and 5% at the retention times of the analytes and internal standards, respectively [24].

Selectivity, sensitivity, and suitability of the assay
The ability of the method to accurately measure analyte concentrations in the presence of endogenous plasma components and exogenous interfering substances was evaluated.Selectivity was assessed by analyzing 8 different lots of drug-free human plasma along with 2 lots each of hemolyzed plasma and lipemic plasma that were processed using the sample preparation method [3].The effect of co-administered medications was studied by spiking acetaminophen, ibuprofen, ranitidine, and ondansetron into analytefree plasma at concentrations equivalent to their reported maximum plasma levels found in patients [21].Any interfering peaks at the retention times of tenofovir, emtricitabine and their internal standards were monitored to determine the selectivity of the method for the intended purpose in real patient samples.

Evaluation of matrix effects
Matrix effects should be evaluated for mass spectrometry-based methods to assess signal suppression or enhancement due to the ionization of analytes in biological matrices.To determine if the method was free from matrix effects, the post-extraction responses of tenofovir and emtricitabine in plasma samples from 10 different donors (including 2 each of hemolyzed and lipemic samples) were compared to the responses in aqueous solutions [25].Matrix effects were determined at the low and high-quality control levels by calculating the matrix factor for the analyte and corresponding internal standard.Then, the matrix factor of the analyte was normalized to the internal standard matrix factor to obtain the internal standard normalized matrix factor.A value of 1 indicates no matrix effects, while the limits for the normalized matrix factor are 0.85 to 1.15 [26].

Method linearity
Three calibration curves were analyzed to evaluate linearity.The peak area ratios of the analyte to standard internal response were plotted on the y-axis against concentration on the x-axis [14,27].

Extraction efficiency (Recovery)
Consistent and comparable extraction recovery during sample preparation was necessary for precise and accurate quantitative results.The relative recovery of tenofovir and emtricitabine was evaluated at three concentration levels (low, medium, and high-quality control samples) by comparing the response of post-extraction spiked plasma samples to pre-extraction spiked plasma samples (n = 6) [14].
Absolute recovery was also determined by comparing the response of extracted plasma samples to standard aqueous solutions [28].

Accuracy and precision
The accuracy and precision of the method over a complete run size were evaluated to demonstrate the reproducibility and consistency of the method [29].A total of 50 replicates each of low, medium, and high-quality control samples were prepared and quantified against freshly prepared calibration curves.The quality control samples were injected in ascending order of concentration from low to high [30].

Stability evaluation
The stability of tenofovir and emtricitabine was assessed under various conditions: short-term bench-top stability at room temperature, freeze-thaw stability after 5 cycles, autosampler stability, dried extract stability, and long-term stability at-70 °C [31].

Method development
To obtain precise and reliable quantification results, it is vital to finetune the chromatographic process variables, sample extraction process, and mass spectrometric parameters with equal importance.These different aspects must be tuned and developed in harmony for the best overall performance of the analytical method [32].

Optimization of LC-MS/MS conditions
The analytes (tenofovir, emtricitabine, and their internal standards) were individually infused into the mass spectrometer to optimize the MS conditions for better sensitivity [33].Initially, a Q1 scan was performed by optimizing the Declustering Potential (DP) and Entrance Potential (EP) to select the parent ion.This was followed by an MS/MS scan to optimize the Collison Energy (CE) and Collison Cell Exit Potential (CXP) to select product ions.In optimizing the mass spectrometric parameters for quantification, the [M + H] + precursor ions observed in the mass spectrum, along with their reproducible fragment ions, were selected for MRM quantification.Unit resolution mode with 300 ms dwell time was employed for every transition.Different stationary phases like C18 and C8 were evaluated.The Phenomenex PFP column gave a better peak shape, resolution, and required sensitivity.A moderate-strength buffer of 5 mmol ammonium formate provided a more signal-to-noise ratio.The total run time was 3.2 min.The product ion spectra of analytes and internal standards are shown in fig. 1.

Sample preparation method development
Both liquid-liquid extraction and solid-phase extraction techniques were evaluated for preparing plasma samples.After considering various methods for solid phase extraction, the Phenomenex Strata-X cartridges were chosen as the preferred option due to their ability to provide high and uniform extraction recoveries, minimal matrix effect, and clear sample extracts.
Although washing the cartridges with 5% methanol initially provided chromatograms with clean peaks, the recovery rates for emtricitabine were observed to be low.Washing the cartridges with 0.1% formic acid solution improved the recovery of emtricitabine.The extraction procedure was carefully optimized and adjusted to obtain similar recoveries for both the analytes (tenofovir and emtricitabine) and their corresponding internal standards (tenofovir-d7 and emtricitabine-d2).The comparable recoveries for analytes and internal standards, along with acceptable normalized response factors of the internal standards, led to a stable and reproducible quantification method [23].

System suitability and repeatability assessment
The actual % CV values found for the area ratios ranged from 0.42% to 2.38% across the entire method validation, demonstrating that the LC-MS/MS system and method yielded repeatable and reproducible results with good precision [21,34].This indicated that the system and analytical method were suitable for the intended purpose.The results of the system suitability and repeatability assessment are shown in table 1.

Carryover and cleaning validation
The results in table 2 show that the carryover is not observed as the peak area in blanks after the Upper Limit of quantification (ULOQ) samples was<20% of LLOQ, indicating good autosampler performance.No interfering peaks were observed in blank water, methanol, or plasma washes, demonstrating the adequacy of the glassware cleaning procedure used [7].

Selectivity, sensitivity, and suitability of the assay
The interference observed in the blank plasma lots was compared to the mean response of processed samples at LLOQ.The precision and accuracy at the LLOQ level were found to be 10.3% and 109.7% for tenofovir and 8.9% and 102.8% for emtricitabine, respectively.Assay suitability was also assessed, and no cross-talk was observed between the analytes and internal standards, demonstrating the specificity of the method [30].

Evaluation of matrix effect
Matrix factor values close to 1 indicate little matrix effect.The average matrix factor values for tenofovir and emtricitabine were close to 1, ranging from 0.992 to 0.975 and 0.946 to 0.972, respectively (results shown in table 3).This suggests a minimal matrix effect.The %CV (coefficient of variation) values were all below 5%, meeting the typical acceptance criteria for matrix effect evaluation.This indicates good precision and reproducibility in the matrix effect assessment.The matrix factor values were determined for various plasma batches, including hemolytic and lipemic samples.The fact that the matrix factor was consistent across different plasma types indicates the robustness of the method and its suitability for real patient samples.Standard deviation and % CV were slightly higher for HQC samples compared to LQC samples.This is not unexpected as the matrix effect and ion suppression tend to increase at higher analyte concentrations [27].

Method linearity
The calibration curves were linear over the range of 5-500 ng/ml for tenofovir and 8-4000 ng/ml for emtricitabine.The slope values were consistent, and correlation coefficients were ≥ 0.99.The mean measured concentrations of the calibration standards met the acceptance criteria of accuracy (±15%) and precision ( ≤15%).Table 4 shows the mean back-calculated concentrations of the calibration standards for tenofovir and emtricitabine.
The accuracy and precision results obtained from dilution integrity experiments at 1:2 and 1:4 dilutions were also within acceptable limits.The blank plasma chromatograms are shown in fig.2, while the chromatograms of the analytes at LLOQC and HQC levels are shown in fig. 3 and 4, respectively.The % mean stability of the analytes ranged from 92.2% to 107.1%.Stability samples at low and high-quality control concentrations were analyzed using a freshly prepared calibration curve.Tenofovir and emtricitabine were found to be stable in human plasma for approximately 16 h at room temperature and after undergoing 5 complete freeze-thaw cycles.The established autosampler stability time (after sample preparation but before injection) was 41 h for tenofovir and emtricitabine, while the dried extract stability (after reconstitution of dried extracts) was 52 h.
The analytes were stable in whole blood for 2.5 h.Long-term stability was evaluated by storing prepared plasma samples at -70 °C, and tenofovir and emtricitabine were found to be stable for at least 32 d under these conditions.

CONCLUSION
A faster and more sensitive LC-MS/MS method was developed for quantifying tenofovir and emtricitabine in human plasma using positive electrospray ionization.The method was validated according to Food and Drug Administration (FDA), Brazilian Health Regulatory Agency, and European Medical Agency guidelines and has a shorter run time of 3.2 min.MRM analysis with optimized MS/MS conditions was used, and solid phase extraction and a Phenomenex Kinetex PFP column were used for separation.No significant interference was observed in blank plasma samples.Thus, this method can be used as a reliable and efficient tool for therapeutic drug monitoring and pharmacokinetic studies of antiretroviral therapy.