HPLC-MS/MS method development for the quantitative determination of nifedipine for Caco-2 permeability assay

Aim. Poorly water-soluble drugs such as nifedipineoffer challenging problems in drug formulation as poor solubility is generally associated with poor dissolution characteristics and thus with poor oral bioavailability (BCS class II drugs). Methods of quantitative determination of nifedipine by methods of spectrophotometry and chromatography are described in the scientific literature. However, methods are not developedfor examination of nifedipine from Caco-2 cell monolayers. Caco-2 cell monolayers have been extensively used for years as a tool to test permeability, assess the oral absorption potential and study the absorption mechanism of compounds. Therefore, the aim of this study was to develop and validate an efficient HPLC MS/MS method for determination of nifedipine from Caco-2 cell monolayers. Materials and methods. Chromatography was achieved on DiscoveryC18, 50 × 2.1 mm, 5 μm column. Samples 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 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, nifedipine was eluted at 1.83 min. A linear response function was established at 1 – 100 ng/mL. The regression equation for the analysis was Y = 0.0323x-0.00121 with coefficient of correction (R2) = 0.9987. According to the Caco2 test results, nifedipine showed high permeability. The within-run coefficients of variation ranged between 0.331% and 0.619% for nifedipine. The within-run percentages of nominal concentrations ranged between 98.80% and 100.63% for nifedipine. The between-run coefficients of variation ranged between 0.332% and 0.615% for nifedipine. The between-run percentages of nominal concentrations ranged between 98.98% and 101.71% for nifedipine. The assay values on both the occasions (intraand inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of nifedipine from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of nifedipine from Caco-2 cell monolayers.


Introduction
Poorly water-soluble drugs such as nifedipine (~ 20 pg/ mL) offer challenging problems in drug formulation as poor solubility is generally associated with poor dissolution characteristics and thus with poor oral bioavailability (BCS class II drugs). Nifedipine is a dihydropyridine calcium channel blocking agent. Nifedipine inhibits the transmembrane influx of extracellular calcium ions into myocardial and vascular smooth muscle cells, causing dilatation of the main coronary and systemic arteries and decreasing myocardial contractility. Chemical name of nifedipine is dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (Fig. 1).
The State Pharmacopoeia of Ukraine (SPhU) (The State Pharmacopeia of Ukraine 2015) has a monograph on the substance of nifedipine. For identification of the substance of nifedipine, the SPhU offers the determination of melting point, absorption spectrophotometry in the infrared region, TLC (mobile phase -ethyl acetate P -cyclohexane (40:60)) and qualitative reaction of formation of azo dye after the previous restoration of nitro group to amino group, quantitative determination -cerimetry. The United States Pharmacopoeia regulates the definition of nifedipine in substances and tablets. For identification, the definition of absorption spectrophotometry in the infrared region and UV-spectrophotometry is proposed. For quantitative determination of nifedipine in tablets -HPLC/UV method. In accordance with this article, the following chromatographic conditions are used: chromatographic column of category L1 (with fixed phase C18) with sixe 4.6 mm x 250 mm; mobile phase -acetonitrile: methanol: water (25:25:50); solvent -methanol, wavelength -235 nm, flow rate -1.0 ml/ min.
The European Pharmacopoeia has (European Pharmacopoeia 2016) a monograph on the substance of nifedipine. For identification, it is proposed to determine the melting point, absorption spectrophotometry in the infrared region, TLC (mobile phase -a mixture of ethyl acetate P and cyclohexane P (40:60 V/V) and qualitative reaction to the primary aromatic amino group -reaction of formation of azo dye (after preliminary reduction of nitro group to amino group). For the quantitative determination of nifedipine -method of cerimetry.
Methods of quantitative determination of nifedipine by methods of spectrophotometry and chromatography are described in the scientific literature (Kondratova et al. 2016(Kondratova et al. , 2017Liliya et al. 2016;Logoyda 2018aLogoyda , b, c, 2019Logoyda et al. 2017aLogoyda et al. , b, c, 2018aMykhalkiv et al. 2018a, b). However, methods are not developedfor examination of nifedipine from Caco-2 cell monolayers (Fujikawa et al. 2005;Gertz al. 2010). Caco-2 cell monolayers have been extensively used for years as a tool to test permeability, assess the oral absorption potential and study the absorption mechanism of compounds (Gozalbes et al. 2011;Hou et al. 2004). Therefore, the aim of this study was to develop and validate an efficient LC MS/MS method for determination of nifedipine from Caco-2 cell monolayers.

Chemicals and reagents
In the present work we used Trypsin EDTA (10×) 0.5% / 0. Nifedipine (purity 99.98%) was purchased from Moehs Catalana, S.L., Spain.Test compound was provided as dry powder (nifedipine) and was dissolved in DMSO at 10 mM to prepare working stocks.

Instrumentation and chromatographic conditions
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, 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. 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 nifedipine. Various analytical columns were tested to obtained good and reproducible response within short run time. The HPLC system was coupled with tandem mass spectrometer API 3000 (PE Sciex). The TurboIonSpray ion source was used in both positive and negative ion modes. Parametrs of electrospray ionizer and MRM characteristics are listed in Table 1. Acquisition and analysis of the data were performed using Analyst 1.5.2 software (PE Sciex).Chromatography was achieved on DiscoveryC18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile -waterformic 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 1.01 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 sample volume was 5 μl.
Caco-2 cells were cultivated in 75 cm 2 flasks to 70-80% of confluence according to the ATCC and Millipore recommendations in humidified atmosphere at 37 °C and 5% CO 2 . Cells were detached with Trypsin/EDTA solution and resuspended in the cell culture medium to a final concentration of 2×10 5 cells/ml. 500 µl of the cell suspension was added to each well of HTS 24-Multiwell Insert System and 35 ml of prewarmed complete medium was added to the feeder tray. Caco-2 cells were incubated in Multiwell Insert System for 21 days before the transport experiments. The medium in filter plate and feeder tray was changed every other day. After 21 days of cell growth, the integrity of the monolayer was verified by measuring the transepithelial electrical resistance (TEER) for every well using the Millicell-ERS system ohm meter. The final TEER values were within the range 150-600 Ω×cm 2 as required for the assay conditions. 24-well insert plate was removed from its feeder plate and placed in a new sterile 24-well transport analysis plate. The medium was aspirated and inserts washed with PBS twice.
To determine the rate of compounds transport in apical (A) to basolateral (B) direction, 300 µL of the test compound dissolved in transport buffer at 10 µM (HBSS, 10 mM HEPES, pH = 7.4) was added into the filter wells; 1000 µL of buffer (HBSS, 10 mM HEPES, pH = 7.4) was added to transport analysis plate wells. The plates were incubated for 90 min at 37 °C with shaking at 100 RPM. 75 µL aliquots were taken from the donor and receiver compartments for LC-MS/MS analysis. All samples were mixed with 2 volumes of acetonitrile with following protein sedimentation by centrifuging at 10000 rpm for 10 minutes. Supernatants were analyzed using the HPLC system coupled with tandem mass spectrometer.
Propranolol (high permeability), Atenolol (low permeability) and Quinidine (moderate permeability) were used as reference compounds.
The apparent permeability (P app ) was calculated for Caco-2 permeability assay using the following equation: P app is expressed in 10 -6 cm/sec. The % recovery can be useful in interpreting the Caco-2 data. If the recovery is very low, this may indicate problems with poor solubility, binding of the compound to the test plate materials, metabolism by the Caco-2 cells or accumulation of the compound in the cell monolayer. The % recovery was calculated using the following equation: V acc -volume of compound solution in acceptor well (cm 2 ), V d -volume of compound solution in donor well (cm 2 ), C acc -concentration of test compound in acceptor well (µM), C initial,d -initial concentration of test compound in a donor well (µM).

Results and discussion
In the present study, optimization and critical evaluation of mobile phase composition, 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. The resolution of peaks was best achieved with DiscoveryC18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile -waterformic 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 1.01 min returns to the initial 0%. Gradient curve shown in Fig. 2. 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.The optimum chromatographic conditions and system suitability parameters are tabulated in Table 2. Nifedipine eluted at ~1.83 minutes. Typical multiple reaction monitoring chromatograms of nifedipineare shown in Fig. 3. A-B permeability data for the test compound of nifedipineand 3 reference compounds are listed in the Table 3. A-B permeability data for all the reference compounds correspond to the literature data, thus validating this study. According to the Caco-2 test results, nifedipineshowed high permeability. It should be noted that the recovery value (Table 4) for nifedipineis 103.74%. On the basis of the data presented in Tables 3, 4 nifedipine can be considered as a highly permeable drug substance. Permeability values obtained in vivo by the intestinal perfusion technique were comparable with the P eff obtained by Caco-2 cell line studies. Permeability values of nifedipine, obtained from a correlation of partition coefficients versus intestinal permeability, also suggest a high permability of nifedipine.
The within-run coefficients of variation ranged between 0.331% and 0.619% for nifedipine. The within-run percentages of nominal concentrations ranged between 98.80% and 100.63% for nifedipine. The between-run coefficients of variation ranged between 0.332% and 0.615% for nifedipine. The between-run percentages of nominal concentrations ranged between 98.98% and 101.71% for nifedipine. Results are presented in Table 5. The assay values on both the occasions (intra-and inter-day) were found to be within the accepted limits.
The results were found to be within the assay variability limits during the entire process.
We selected nifedipine as a CYP3A4 test drug because in vitro results were obtained by the closely related nifedipine derivative denitronifedipine. Nifedipine undergoes extensive CYP3A4-dependent biotransformation both in the gut wall and the liver. When the criteria of the Guidances are strictly applied, nifedipine is a BCS Class II substance and this API can not be considered a candidate for granting a biowaiver. From a scientific point of view,nifedipine is a candidate for granting a biowaiver when the tablets are formulated with well-known excipients, show rapid in vitro dissolution, and meet the dissolution profile comparison criteria as defined in the Guidances, but with a redefined upper boundary for the pH of 6.8. The USP criteria and method are suitable to assure batch to batch consistency.
In summary it can be concluded that developed method is simple and rapid for determination of nifedipine from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of nifedipine from Caco-2 cell monolayers.