Bioanalytical method development and validation for the determination of metoprolol and meldonium in human plasma

Aim.The main purpose of this study was to develop a simple, precise, rapid and accurate method for the quantification of metoprolol and meldonium in human plasma. Materials and methods. The resolution of peaks of metoprolol was best achieved with Discovery C18, 50 × 2.1 mm, 5 μm column and meldonium ZORBAX HILIC Plus, 50 × 2.1 mm, 3.5 μm column. Samples of metoprolol were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.11 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 mL/min into the mass spectrometer ESI chamber. The injection volume was 5μl. Samples of meldonium were chromatographed in a isocratic using mobile phase water – acetonitrile – ammonium formate buffer 200 мМ, 20 : 75 : 5 v/v). Results.The total chromatographic run time was 2.0 minutes and the elution of metoprolol, meldonium and IS occurred at ~1.39 and 1.18 minutes, respectively.A linear response function was established at 2 200 ng/mL for metoprolol and 50 -5000 ng/mL for meldonium in human plasma. The% mean recovery for metoprolol in LQC, MQC and HQC was 99.0%, 107.5% and 96.8%, for meldonium in LQC, MQC and HQC was 94.1%, 100.2% and 93.1% respectively. The lowest concentration with the RSD <20% was taken as LLOQ and was found to be 2.31 ng/mL for metoprolol, 47.70 ng/mL for meldonium. The % accuracy of LLOQ samples prepared with the different biological matrix lots were found 115.4% for metoprolol and 95.5% for meldonium, which were found within the range of 80.00–120.00% for the seven different plasma lots. % CV for LLOQ samples was observed as 12.8% and 7.7% respectively, which are within 20.00% of the acceptance criteria. Conclusion.A rapid method was developed for simultaneous determination of metoprolol and meldonium in human plasma. The method was strictly validated according to the ICH guidelines. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for routine examination of metoprolol and meldonium in human plasma.


Introduction
Metoprolol (Fig. 1) is a beta1-selective (cardioselective) adrenoceptor blocking agent, for oral administration, available as extended-release tablets. Metoprolol succinate extended-release tablet has been formulated to provide a controlled and predictable release of metoprolol for once-daily administration. The tablets comprise a multiple unit system containing metoprolol succinate in a multitude of controlled release pellets. Each pellet acts as a separate drug delivery unit and is designed to deliver metoprolol continuously over the dosage interval. The tablets contain 23.75, 47.5, 95 and 190 mg of metoprolol succinate equivalent to 25, 50, 100 and 200 mg of metoprolol tartrate, USP, respectively. Its chemical name is (±)1-(isopropylamino)-3-[p-(2-methoxyethyl) phenoxy]-2-propanol. Metoprolol is a propanolamine that is 1-(propan-2-ylamino) propan-2-ol substituted by a 4-(2-methoxyethyl)phenoxy group at position 1. It has a role as a beta-adrenergic antagonist, an antihypertensive agent, a xenobiotic and an environmental contaminant. It is a propanolamine, an aromatic ether, a secondary alcohol and a secondary amino compound. Metoprolol is used for a number of conditions, including hypertension, angina, acute myocardial infarction, upraventricular tachycardia, ventricular tachycardia, congestive heart failure, and prevention of migraine headaches.
The State Pharmacopoeia of Ukraine (SPhU) does not have a monograph on the substance of metoprolol or on the prepared medical form (The State Pharmacopeia of Ukraine 2015). However, the United States Pharmacopoeia regulates the determination of metoprolol succinate in extended-release tablets. For identification, IR-spectroscopy and HPLC are proposed. For quantitative determination of metoprolol succinate in tablets in assay and dissolution test -HPLC, respectively. Chromatographic conditions for the determination of metoprolol succinate, tablets are given in the monograph of the United States Pharmacopoeia, which is used the chromatographic column 4-mm × 12.5-cm; 5-µm packing L7 and mobile phase consisting of acetonitrile and buffer (25:75). Mobile phase rate -1 ml/min, detection wavelength -280 nm, tailing factor -NMT 2.0, relative standard deviation -NMT 2.0%. The proposed method of the United States Pharmacopeia requires a long sampling.
The European Pharmacopeia has a monograph on metoprolol tartrate tablets. Identification of metoprolol tartrate of the European Pharmacopeia regulates the absorption spectrophotometry in the infrared region, UV-spectrophotometry and HPLC, quantitative determination -HPLC/UV (European Pharmacopoeia 2016). As a solvent, mixture of methanol and 0.1 M hydrochloric acid is used, mobile phase -solution of 1-pentanesulfonic acid sodium salt (monohydrate), anhydrous sodium acetate in mixture of methanol and water and adding glacial acetic acid. Mobile phase rate -1.0 ml/min, detection wavelength -254 nm.
Nowadays metabolic therapy is an important component of the treatment of virtually any disease of the internal organs. Drugs affecting the metabolic processes in the heart, brain, liver, muscles, are widely prescribed by general practitioners and narrow specialists. A special place among them take cardioprotectors -a group of drugs that improves metabolic processes in ischemic myocardium, increase resistance to hypoxia, eliminate cellular metabolism disorders. To the well-known and recognized by clinicians cardioprotectors also belongs meldonium Meldonium, sold under the brand name Mildronate, is a performance-enhancing drug that is the source of much debate in the world of sports doping. Initially designed in Latvia for use in animals, the World Anti-Doping Agency (WADA) has indicated that this drug is banned in Olympic sports. Meldonium is primarily manufactured by a Latvian drug company called Grindeks. It is also known as Mildronāts, Quaterine, trimethylhydrazinium propionate, and 3-(2,2,2-trimethylhydraziniumyl)propionate (Fig. 2). Meldonium is a structural analogue of gamma-butyrobetamine. At the heart of the mechanism of action of the drug is the reduction in the amount of carnitine in the body, which in the conditions of oxygen insufficiency leads to inhibition of oxidation of fatty acids. Meldonium induces biosynthesis of nitric oxide, which promotes relaxation of smooth muscle of vessels, improves microcirculation and endothelial function. The therapeutic effect of the drug is due to the variety of its pharmacological effects. Meldoniumhas been classified as a hydrazinium base of low molecular weight, and its determination is rather difficult. The State Pharmacopoeia of Ukraine (SPhU) does not have a monograph on the substance of meldonium dihydrate or on the prepared medical form (The State Pharmacopeia of Ukraine 2015). However, the substance meldoniumdihydrateis described in the European Pharmacopoeia and the State Pharmacopoeia of the Russian Federation (SPRF). Thus, the SPRF XII recommends the quantitative determination of meldoniumdihydrate by the acidimetry method   in a non-aqueous medium with a potentiometric fixation of the end point of titration. Combination theraphy of meldonium and metoprolol is used in the treatment of various chronic cardio-vascular diseases and disorders of the cerebral circulation, as well as to improve mental and physical capacity. In the contemporary literature, some bioanalytical methods were reported for quantification of metoprolol and meldonium in human plasma (Albers et al. 2005;Braza et al. 2002;Angier et al. 2005;Jensen et al. 2008;Gowda et al. 2007;Peng et al. 2010;Zupanets et al. 2010;Kondratova et al. 2016Kondratova et al. , 2017Liliya et al. 2016;Logoyda 2018aLogoyda , b, c, 2019Logoyda et al. 2017aLogoyda et al. , b, c, 2018aMykhalkiv et al 2018a, b). Therefore, it was thought desirable to develop a simple, accurate, cheap and fast procedure that could be applied for the determination of metoprolol and meldonium in human plasma, this study performed assay validations as per guidelines. While this method with validation details were economical and applied for pharmacokinetic studies of metoprolol and meldonium.

Standard solutions
Metoprolol, meldonium and IS were weighed accurately into volumetric flasks using an analytical microbalance. Approximately 0.5 mg/mL primary stock solutions of metoprolol, 2.5 mg/ml primary stock solutions of meldonium, 1.0 mg/mL primary stock solutions of bethanechol (IS), 0.2 mg/mL primary stock solutions of bisoprolol (IS) were prepared in methanol. The stock solutions were stored at −20 °C, which were found to be stable for 1 month. The stock solutions of metoprolol and meldonium were successively diluted with methanol and water to prepare secondary stocks and working solutions. Secondary stock solutions and working solutions were used to prepare calibration curve (CC) and quality control (QC) samples. Working stock solutions were stored at 4 °C for a week. Working stocks were used to prepare plasma calibration standards. Blank human plasma was screened before spiking to ensure that it was free from endogenous interference at retention times of metoprolol, meldonium and IS, respectively. Calibration standards' samples were pre-

Sample preparation
A simple protein precipitation extraction method was followed for extraction of metoprolol and meldonium at from human plasma. From the deep freezer, the required quantities of CC standards and QC samples were withdrawn. The samples were thawed at room temperature. To an aliquot of 100 μl plasma, 20 μl of IS was added. To this mixture, 300 μl of methanol was added and vortexed for 2 minutes, followed by centrifugation at 6000 rpm for 5 minutes at 4 °C. After centrifugation, approximately 50 μl supernatant was aliquoted into, respectively, labeled autosampler vials, which were later placed in the autosampler at 15 °C ± 4 °C. 10μl of the sample was injected onto LC-MS/MS system for analysis.

Method validation
A full validation according to the ICH guidelines was performed for the assay in K 2 EDTA human plasma (Guideline on Validation of Bioanalytical Methods 2009, Guidance for Industry. Bioanalytical Method Validation. Food and Drug Administration 2010).

Specificity
The specificity of the method was evaluated by analyzing human plasma samples from different lots to investigate the potential interferences at the chromatographic peak region for analytes and IS. The acceptance criterion for the experiment was that should have <20% area response to that of the LLOQ level response in the same matrix. Two lots of hemolyzed plasma samples were also analyzed to ensure specificity against potential biological interferences.

Linearity
The points CC (2-200 ng/ml for metoprolol and 50-5000 ng/ml for meldonium) were constructed by plotting the peak area ratio of each analyte: IS against the nominal concentration of calibration standards in K 2 EDTA human plasma. Following the evaluation of different weighing factors, the results were fit into linear regression analysis using 1/X2 (X: Concentration) weighing factor. The CC should have a correlation coefficient (r) of 0.99 or better. The acceptance criteria for each back-calculated standard concentration were ±15% deviation from the nominal value except at LLOQ, which was set at ±20%.

Matrix effect
The effect of human plasma constituents over the ionization of metoprolol, meldonium and IS was determined by post-column infusion method to evaluate matrix effect. Briefly, an infusion pump delivers a constant amount of analyte into LC system outlet entering to mass spectrometer inlet. To follow the analyte signal, the mass spectrometer was operated in MRM mode. The human plasma constituent sample extract was injected on LC column. A steady ion response was obtained as a function of time since the analyte was infused at a constant rate. Any endogenous compound that elutes from the column which causes a variation in ESI response of the infused analyte was seen as a suppression or enhancement in the response of the infused analyte. A separate experiment was performed with metoprolol, meldonium and IS solutions, which were infused at a constant rate, and blank matrix sample injected through the LC. To evaluate matrix effect, different lots of human plasma were spiked with analyte concentration levels at LQC and HQC levels. According to guidelines, the acceptance criterion for each back-calculated concentration was ±15% deviation from the nominal value.

Precision and accuracy
The intra-assay precision and accuracy were estimated by analyzing six replicates containing metoprolol, meldonium at four different QC levels concentrations (

Recovery
The efficiency of metoprolol, meldonium and IS extraction from human plasma was determined by comparing the responses of the analytes extracted from replicate QC samples (n = 6) with those of neat standard solutions spiked in post-extracted plasma blank sample at equivalent concentrations by protein precipitation extraction method. Recovery of metoprolol was determined at LQC (5.94 ng/mL) and HQC (145 ng/mL) concentrations, meldonium was determined at LQC (141 ng/mL) and HQC (3492 ng/mL) concentrations whereas the recovery of IS was determined at a single concentration of 20 ng/mL.

Stability experiments
Stability tests were conducted to evaluate the stability of metoprolol, meldonium in plasma samples under different conditions. 8 hrs bench top stability, processed samples stability (autosampler stability for 26 hrs at 10 °C), three cycles of freeze-thaw stability, 30 days of long-term stability at −80±10 °C were performed at LQC and HQC levels using six replicates at each level. Samples were considered stable if assay values' acceptance criterion was of accuracy (i.e., 85-115% from fresh samples) and precision (i.e., ±15% relative standard deviation [RSD]).

Results and discussion
Meldonium can be perfectly determined by HILIC chromatography and metoprolol by conventional reversed-phase HPLC. We tried to find a common method that would allow us to identify both analytes at one time -reversed-phase HPLC with an ion-pair reagent for meldonium. Alas, such a mutual compromise struck on both analytes, and failed to obtain the necessary results, despite several attempts, even when switching to a more sensitive instrument. As a result, we had to go back to the separate methods on the surface, and immediately everything turned out. Sampling remained common -methanol precipitation. In the present study, optimization and critical evaluation of mobile phase composition (gradient), flow rate, and analytical column were important to obtain good resolution of peaks of interest from the endogenous components, which in turn affect reproducibility and sensitivity of the method. Selection of chromatographic conditions for the proposed method was optimized to suit the preclinical pharmacokinetic studies. To ease the sample preparation in microtubes and to reduce the usage of solvent, the plasma volume was kept low. Initial feasibility experiments of a various mixture(s) of solvents such as acetonitrile, methanol and formic acid along with altered flow rates (in the range of 0.1-0.6 ml/min) were performed to optimize an effective chromatographic resolution of metorolol, meldonium and IS. Various analytical columns were tested to obtained good and reproducible response within short run time. The resolution of peaks of metoprolol was best achieved with DiscoveryC18, 50 × 2.1 mm, 5 μm column and meldonium -ZORBAX HILIC Plus, 50 × 2.1 mm, 3.5 μm column. Samples of metoprolol were chromatographed in a gradient mode (eluent A (acetonitrile -water -formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile -formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.11 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 mL/min into the mass spectrometer ESI chamber. The injection volume was 5 μl. Samples of meldonium were chromatographed in a isocratic using mobile phase water -acetonitrile -ammonium formate buffer 200 мМ, 20 : 75 : 5 v/v).
The purpose of sample extraction optimization is mainly to achieve high extraction recovery with negligible or low matrix effects to improve sensitivity and reliability of LC-MS/MS analysis. A poor extraction procedure decreases method robustness due to the presence of endogenous interference in the sample extracts, which are not efficiently cleaned up due to poor extraction procedure decreases the method robustness due to the endogenous interference in the sample extracts. With time-saving advantage and simplicity, the protein precipitation extraction method was chosen as an extraction method. The attained LLOQ was sufficient to quantify metoprolol and meldonium in low-dose pharmacokinetic studies according to Guideline EMEA/CHMP and Guideline FDA/CDER.
Metoprolol and meldonium eluted at ~1.39 and 1.18minutes, respectively. During a direct infusion experiment, the mass spectra for metoprolol, meldonium and IS revealed peaks at m/z 347.128, 377.165 and 455.385, respectively as protonated molecular ions, [M+H] + . Typical multiple reaction monitoring chromatograms of metoprolol, meldonium and internal standard in dipotassium ethylenediaminetetraacetic acid human blank plasma are shown in Figs 3, 4.
The total chromatographic run time was 2.0 minutes and the elution of metoprolol and meldonium occurred at ~1.39 and 1.18 minutes, respectively.

Specificity
Different lots of plasma were analysed to ensure that no endogenous interferences were present at the retention time of metoprolol and meldonium LLOQ level samples along with plasma blank from the respective plasma lots were prepared and analysed (Table 3).
In all plasma blanks, the response at the retention time of metoprolol and meldonium was less than 20% of LLOQ response and at the retention time of IS, the response was less than 5% of mean IS response in LLOQ.

Linearity
The calibration standard curves had a reliable reproducibility over the standard concentrations across the calibra-     tion range. The average regression (n = 3) was found to be > 0.997 for all analytes. The calibration curve (peak area ratio Vs Concentration) was linear over working range for metoprolol of 2 to 200.00 ng/mL with 7 point calibration used for quantification by linear regression, shown in Fig. 5. The regression equation for the analysis was Y = 0.00406x + 0.00381 with coefficient of correction (R 2 ) = 0.9990.
The calibration curve (peak area ratio Vs Concentration) was linear over working range for meldonium of 50 to 5000.00 ng/mL with 7 point calibration used for quantification by linear regression, shown in Fig. 6. The regression equation for the analysis was Y = 0.000136x + 0.000715 with coefficient of correction (R 2 ) = 0.9975.

Intraday (within run) and Inter-day (between run) precision and accuracy
The within-run coefficients of variation ranged between 0.331% and 0.619% for metoprolol. The within-run percentages of nominal concentrations ranged between 98.80% and 100.63% formetoprolol. The between-run coefficients of variation ranged between 0.332% and 0.615% for metoprolol. The between-run percentages of nominal concentrations ranged between 98.98% and 101.71% for metoprolol. Results are presented in Table  6. The assay values on both the occasions (intra-and inter-day) were found to be within the accepted limits.
The within-run coefficients of variation ranged between 0.353% and 0.719% for meldonium. The within-run percentages of nominal concentrations ranged between 99.23% and 101.17% for meldonium. The between-run coefficients of variation ranged between 0.349% and 0.674% for meldonium. The between-run percentages of nominal concentrations ranged between 99.57% and 101.79% for meldonium. Results are presented in Table 7.

Matrix effect
The lowest concentration with the RSD < 0% was taken as LLOQand was found to be 2.31 ng/mL for metoprolol, 47.70 ng/mL for meldonium. The% accuracy of LLOQ samples prepared with the different biological matrix lots were found 115.4% for metoprolol and 95.5% for meldonium, which were found within the range of 80.00-120.00% for the seven different plasma lots.% CV for LLOQ samples was observed as 12.8% and 7.7% respectively, which are within 20.00% of the acceptance criteria. Results are presented in Tables 8, 9.   Table 9. Results of matrix effect of meldonium. Abbreviations: Lower limit of quantification (LLOQ). Each value is represented as a mean ± SD of 5observations (n = 5), SD: Standard Deviation, RSD: Relative Standard Deviation, #Acceptance criteria < 2.0.

Stability
The predicted concentrations for metoprolol (5.94ng/ mL and 145 ng/mL) and meldonium (141 ng/mL and 3492 ng/mL) deviated within ± 15% of the fresh sample concentrations in a battery of stability tests namely, in-injector (22 hrs), bench-top (7 hrs), and repeated four freeze/thaw cycles stability (Table 10). The results were found to be within the assay variability limits during the entire process.

Conclusion
A highly sensitive, specific, reproducible, rapid and high-throughput LC-MS/MS assay was developed and validated to quantify metoprolol and meldonium in human plasma as per the regulatory guidelines. The present method involved a simple precipitation method of sample preparation, which gave consistent and reproducible recoveries.
Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for routine examination of metoprolol and meldonium in human plasma. The combination was taken up for developing a bioanalytical method development and validation so that further it would be useful for performing pharmacokinetic studies.