UHPLC-PDA Assay for Simultaneous Determination of Vitamin D3 and Menaquinone-7 in Pharmaceutical Solid Dosage Formulation

A newly developed method based on ultrahigh performance liquid chromatography (UHPLC) was optimized for the simultaneous determination of vitamin D3 and menaquinone-7 (MK-7) in tablet formulation in the present study. UHPLC separation of vitamin D3 and MK-7 was performed with ACE Excel 2 C18-PFP column (2 μm, 2.1 × 100 mm) at 0.6 mL min−1 flow rate, whereas the mobile phase consisted of methanol/water (19 : 1, v/v, phase A) and isopropyl alcohol (99.9%, phase B) containing 0.5% triethylamine. Isocratic separation of both the analytes was performed at 40°C by pumping the mobile phases A and B in the ratio of 50 : 50 (v/v, pH, 6.0). Both analytes were detected at a wavelength of 265 nm and the injection volume was 1.0 μL. The overall runtime per sample was 4.5 min with retention time of 1.26 and 3.64 min for vitamin D3 and MK-7, respectively. The calibration curve was linear from 5.0 to 100 μg mL−1 for vitamin D3 and MK-7 with a coefficient of determination (R2) ≥ 0.9981, while repeatability and reproducibility (expressed as relative standard deviation) were lower than 1.46 and 2.21%, respectively. The proposed HPLC method was demonstrated to be simple and rapid for the determination of vitamin D3 and MK-7 in tablets.


Introduction
Vitamin D 3 (cholecalciferol) is chemically known as (3 ,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3-ol (C 27 H 44 O, 384.64 g/mol) which belongs to fat soluble secosteroids group. Vitamin D 3 can be ingested from the diet and supplements but it is naturally produced by human body after UVB (280-315 nm) radiation exposure. Active form of vitamin D 3 (1 , 25 (OH) 2 D 3 ) exists in liver and kidney after hydroxylation [1,2]. Vitamin K belongs to class of fat soluble vitamins comprising a number of structurally related compounds including vitamin K1 (phylloquinone) and vitamin K2s (menaquinones). Menaquinone-7 (MK-7) is the most important nutrition and all-trans menaquinone-7 is the active form. MK-7 is chemically known as 2-[(2E,6E, 10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6, 10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-dione (C 46 H 64 O 2 , 649.02 g/mol). The chemical structures for both components are depicted in Figure 1. Menaquinones are produced by bacteria in process of fermentation; however, it can be produced synthetically [3,4]. Imbalance diet and acute or chronic illness are most frequently induced nutritional deficiency. Nutritional deficiency can also be provoked by medical treatment and surgical stress. Nutritional deficiencies, particularly vitamins imbalance, are caused by pharmacological agents like folate antagonists, anticoagulants, diuretics, antacids, oral hypoglycemic agents, antibiotics, anesthetic agents, and psychotropic agents [5]. Nonabsorbed sucrose polyester and drugs that induced changes in lipid processing in the gastrointestinal tract are responsible for deficiencies of lipid soluble vitamins (vitamin D 3 and MK-7). Vitamins D 3 and K deficiencies occur due to induction of Cyp450 and eradication of bacteria (responsible for synthesis of vitamin K), respectively [6][7][8]. Many scientific papers described the analysis of vitamin D 3 and MK-7 in various matrixes such as biological fluids, foods, plant material by fluorimetric, UV-Vis, and MS detection after chromatographic separation [9][10][11][12][13][14]. Determination of vitamin K is also reported by HPLC coupled to chemiluminescence and electrochemical detectors [15,16]. Using the above-mentioned techniques, a good sensitivity can be achieved but cost and complexity associated with these methods are problematic for routine analysis in quality control (QC) laboratories of pharmaceutical industries. So we need to develop a rapid and cost effective method for routine analysis of vitamin D 3 and MK-7 in QC laboratories. The proposed method describes the optimization of UHPLC method and its comparison with HPLC. HPLC is prominent technique in laboratories for the last 30 years but did it not keep the pace with growing demand of analysis in short interval of time with reliability. Using UHPLC, more samples can be analysed in very short time with reliability and accuracy. Eddy and longitudinal diffusion coefficients in Van Deemter equation play important role regarding the separation of analytes. These coefficients are directly related to particle size of column packing and decreased with small particle size which results in better resolution [17][18][19][20]. The goal of present study was to optimize and validate the UHPLC method for simultaneous determination of vitamin D 3 and MK-7 in tablet formulations. The developed method was compared with HPLC to prove its adequacy for pharmaceutical studies with minimum consumption of solvents, high resolution, and symmetrical peaks. ICH (International Council for Harmonization) guidelines [21][22][23][24] were followed to validate the proposed UHPLC method.

Standard and Working Solutions.
Individual stock standard solution of vitamin D 3 and MK-7 (1000 g mL −1 ) was prepared in ethanol in ultrasonic bath for 15 min and working solutions of vitamin D 3 and MK-7 were prepared from stock standard solution in mobile phase. Mixed standard solutions of vitamin D 3 and MK-7 (25.0 g mL −1 each) were also prepared by diluting the stock standard solution in mobile phase.

Analysis of Tablet Formulation by Standard Addition.
The stated composition of tablet (Avelia5) is vitamin D3 (10 g) and MK-7 (90 g) was analysed by the proposed method. Twenty tablets were grinded and aliquots equivalent to one tablet were diluted with ethanol containing 1.24 mg of vitamin D 3 and 1.16 mg of MK-7 in 50 mL flask and final concentration of each analyte became 25 g mL −1 . Then they were sonicated for 15 min for complete dissolution and finally diluted with mobile phase.

Validation Studies.
Validation studies were performed to characterize the proposed analytical method such as specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), and conformity of chromatographic parameters (tailing factor, selectivity factor, resolution, and theoretical plates). Conformity of chromatographic conditions is basically system suitability tests which are foremost part of validation studies. So system suitability tests were performed in a prior step of validation studies.
2.6. Specificity. Analysis of placebo was performed to assess the specificity of the proposed chromatographic method [24,25]. Sodium starch glycolate, magnesium stearate, sodium lauryl sulphate, polyvinyl povidone (PVP-K30), and polyethylene glycol (PEG-6000) were dissolved in ethanol and dilutions were made in mobile phase for specificity studies.

Accuracy and Precision.
The accuracy of each method was determined in triplicate by spiking a known amount of each analyte standard solution in the dosage form (10 g tablet content + 1240 g standard added in 50 mL = 25 g mL −1 vitamin D 3 and 90 g tablet content + 1160 g standard added in 50 mL = 25 g mL −1 ) resulting in final concentrations of 37.5, 50.0, and 62.5 g mL −1 , for vitamin D 3 and MK-7. This represented 50, 100, and 150% of each analyte in the dosing formulation. For precision determination, vitamin D 3 and MK-7 were spiked at 20.0, 25.00, and 30.0 g mL -1 representing 80, 100, and 120% of each analyte, resulting in final concentrations of 45.0, 50.0, and 55.0 g mL −1 , for each analyte. The intraday precision (repeatability) was evaluated by replicates of five on one day, whereas the interday precision (reproducibility) was determined over three consecutive days.

2.9.
Method's LOD/LOQ. Vitamin D 3 and MK-7 standard solution was injected in replicates of six. The resultant parameters of the linear regression including the standard deviation (SD) of the response based upon the slope and intercept determined the LOD and LOQ of the UHPLC method. The LOD and LOQ were defined as 3.3 / and 10 / , respectively [26][27][28][29], where is standard deviation and is slope of regression line.

Method Robustness.
Small but deliberate changes in chromatographic conditions such as mobile phase, pH, column temperature, and flow rate were done to evaluate the robustness of the proposed UHPLC method.

Optimization of Chromatographic Conditions.
Optimization of HPLC and UHPLC method was done by performing system suitability tests; in range of 200-400 nm, both vitamin D 3 and MK-7 were scanned and absorption spectrum was noted. Both analytes were absorbed in this range with absorption maximum at 265 nm. Four different mobile phase ( : ) compositions such as 60 : 40, 50 : 50, 40 : 60, and 30 : 70 were examined to optimize chromatographic conditions such as tailing factor ( ≤ 2), selectivity factor ( > 1), resolution (Rs > 2), and theoretical plates ( > 2000) to get compliance with ICH guidelines (Table 1). For HPLC, these mobile phases were run on different columns like ACE 5 C18, Venusil XBP C18, Hypersil ODS, and Purespher5 RP-18, while ACE Excel 2 C18-PFP, Waters ACQUITY 1.7 BEH C 18, Agilent Poroshell 2.7 120 EC C18, and Phenomenex Kinetex 2.6 C18 were employed with UHPLC at different pH (3.0, 4.0, 5.0, and 6.0). The typical chromatograms of vitamin D 3 and MK-7 with and without placebo obtained by both the LC-based methods are presented in Figures 2 and 3. Free silanol in column packing could be interacting with drugs of both acidic and basic nature. In order to improve the peak shapes, TEA, a silanol blocker, was added to the mobile phase (0.5%, v/v). Silanol blocker provided additional selectivity byand dipole interaction which resulted in achieving the better overall resolution [30].
In the end, the mobile phase consisting of MeOH/H 2 O and IPA in a ratio of 50 : 50 (v/v) with the addition of 0.5% TEA was found to be excellent using ACE 5 C18 and ACE Excel 2 C18-PFP columns for UHPLC and HPLC analysis, respectively. The chromatographic parameters under final conditions are summarized in Table 2 exhibiting an excellent peak shape, resolution, and higher number of theoretical plates.

Validation Studies.
The specificity of the optimized UHPLC method was examined with vitamin D 3 and MK-7 at concentration each of 25.00 g mL -1 , relative to the blank mobile phase (Figure 3). The presence of placebo did not interfere during the determination of vitamin D 3 and MK-7 as the components were baseline separated. For both chromatographic methods over a dynamic range of 5.0-100 g mL -1 , seven concentrations (5.0, 10.0, 15.0, 25, 50.0, 75.0, and 100 g mL −1 ) were employed to construct a calibration graph for vitamin D 3 and MK-7. The calibration curves were linear for vitamin D 3 and MK-7 with a coefficient of determination ( 2 ) ≥ 0.9981 regardless of the LC-based    Table 3). Accuracy of methods by both the techniques under investigation was performed by evaluating the recovery studies after spiking the known amount of standard drugs in commercial products. LOD and LOQ determined by UHPLC were 1.5-fold less than HPLC which is due to much sensitive detector -DReC employed in UHPLC. The recovery results were obtained between the ranges of 98.97-101.74% and 99.36-101.56% (Table 4) for HPLC and UHPLC, respectively, which justified the suitability of the techniques for their intended applications. In addition, the results obtained were not differing significantly among the tested methods (HPLC and UHPLC) employed for determination of vitamin D 3 and MK-7 at 95% of confidence interval. All the experimental -values andvalues (Table 4) were below the theoretical -values (4.30) and -values (19.0). Advantages of UHPLC over HPLC were its rapidity, ease of operation, high selectivity, and consumption of minimum amount of solvents. For precision studies, the results of repeatability and reproducibility are presented in Table 5 by injecting three different concentrations (80, 100, and 120% level of analyte under investigation) of standard solutions of vitamin D 3 and MK-7 ( = 5) on the same day and three consecutive days, respectively. RSD values for repeatability and reproducibility were obtained less than 1.92 and 2.30, respectively, for HPLC and less than 1.46 for repeatability and 2.21 for reproducibility assays with UHPLC.

Method Robustness.
The robustness of the proposed UHPLC method was evaluated by slight changes of the chromatographic parameters including the flow rate (±0.1 mL min −1 ), mobile phase ratio (±5.0 mL), column     temperature (±5 ∘ C), wavelength (±2 nm), and pH (±0.1). Afterwards, the drug contents besides chromatographic parameters like retention time, tailing factor, number of theoretical plates, and resolution were determined. The results summarized in Table 6 demonstrated that the effects of the deliberate changes in chromatographic conditions are neglectable and that the proposed UHPLC method was robust for its intended applications.

Analysis of Commercial Tablet Formulation.
The applicability of the proposed UHPLC method was evaluated by examining the commercial tablet (Avelia) with reported concentration of vitamin D 3 (10 g) and MK-7 (90 g). Since the tablet contained the microcontents of both the analytes, for better analysis performance and to get reliable assay results, standard addition method was adopted. It was ensured that the removal of the excipients with an extraction step before analysis was unnecessary. It was concluded that the proposed UHPLC method was sufficiently accurate and precise (Table 7) with recovery and RSD found was 103.59, 102.87% and 1.20, 1.12% for vitamin D 3 (10 g) and MK-7 (90 g), respectively.

Conclusion
In the literature, UHPLC method for simultaneous determination of vitamin D 3 and MK-7 in pharmaceutical formulations is not found available. For this reason, UHPLC method was fully validated according to ICH guidelines and was presented for determination of vitamin D 3 and MK-7 in tablet formulations. Remarkable advantages of UHPLC over HPLC were found such as rapidity, ease of operation, high selectivity, and consuming minimum amount of solvents. Good recoveries, interference-free, and high reproducible chromatograms were achieved. The proposed method was optimized step by step and presented its suitability for quality control laboratories where time and economy are essentially required. The proposed method showed its adequacy with high recovery in the presence of excipients and additives used in the formulations.

Disclosure
This article does not contain any studies with human participants or animals performed by any of the authors.