High performance thin-layer and high performance liquid chromatography coupled with photodiode array and fluorescence detectors for analysis of valsartan and sacubitril in their supramolecular complex with quantitation of sacubitril-related substance in raw material and tablets

High performance thin-layer and high performance liquid chromatography coupled with photodiode... Abstract Valsartan (VAL) and sacubitril (SAC) are combined in a supramolecular complex, LCZ696, which is a newly approved remedy for heart failure. SAC-related substance (biphenyl methyl pyrrolidinone [BMP]) which also appears as an intermediate during SAC synthesis is considered to be a suspected impurity for SAC and/or LCZ696 tablets. The study investigates the analysis of VAL and SAC in their supramolecular complex along with SAC-related substance, BMP, using high performance thin-layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) with two different detectors; fluorescence detector (FLD) and diode array detector (DAD). The work aimed at analyzing BMP at low levels in the presence of its parent drug, SAC. BMP was successfully analyzed at a level of 0.167, 1 and 3% of its parent drug, SAC upon using HPLC-FLD, HPLC-DAD and HPTLC, respectively. For HPLC-FLD, the detector was set at λex/λem (nm/nm): 0–4.5 min at 255/374; 4.5–6 min at 255/314, for achieving an adequate sensitivity of the method to monitor and quantify VAL and SAC in the presence of BMP. Low limits of detection (8.3, 3.3 and 1.7 ng mL−1) and limits of quantitation (25, 10 and 5 ng mL−1) values obtained for VAL, SAC and BMP, respectively, upon using FLD suggest that low level of baseline noise enables the detection and quantitation of low BMP concentration. Introduction The new remedy of LCZ696 supramolecular complex is claimed to assist reducing the possibility of cardiovascular mortality and heart failure hospitalization in patients (1). Moreover, it is recommended to be used instead of angiotensin converting enzyme inhibitors or angiotensin II receptor blocker for first-line treatment of these patients (2–4). Valsartan (VAL) is N-pentanoyl-N-{[2′-(1H-tetrazol-5-yl) [1,1′-biphenyl]−4-yl] methyl}-l-valine (Figure 1a). It is mostly used for treatment of high blood pressure and congestive heart failure (5). Different chromatographic methods were found in literature assaying VAL either alone or in combination with other antihypertensive medications including high performance liquid chromatography fluorescence detector (HPLC-FLD) (6,7), high performance thin-layer chromatography (HPTLC) (8–13) and HPLC-diode array detector (HPLC-DAD) (8,14,15). Figure 1. View largeDownload slide Structures of valsartan (a), sacubitril (b), intermediate (c) and their supramolecular complex, LCZ696 (d). Figure 1. View largeDownload slide Structures of valsartan (a), sacubitril (b), intermediate (c) and their supramolecular complex, LCZ696 (d). Sacubitril (SAC) is 4-{[(1 S,3 R)-1-([1,1′-biphenyl]-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl] amino}-4-oxobutanoic acid (Figure 1b). It is a pro-drug, its active metabolite prevents neprilysin which is a neutral endopeptidase (16). So far nothing has been found in literature regarding this novel combination LCZ696 (VAL/SAC) except four works of literature. One of them is reversed phase-HPLC method for their assaying in tablets, another one is LC-MS/MS which deals with their quantification in rat plasma while the most recent two reports are the authors’ works, the first is investigating the spectrofluorimetric behavior of SAC and analyzing it with VAL in LCZ696 using derivative-based spectrofluorimetric method while the second investigates the SAC degradation kinetics with its HPLC analysis in LCZ696. (17–20). SAC-related substance (biphenyl methyl pyrrolidinone [BMP]) is (S)-5-biphenyl-4-ylmethylpyrrolidin-2-one (Figure 1c). It is found in the pathway of synthesis of SAC as intermediate (21,22). So, it is considered to be a suspected impurity that could be found either in SAC raw materials and/or LCZ696 (Figure 1d) tablets. Most active pharmaceutical ingredients (API) are being synthesized by organic chemical syntheses. Various components, for instance: very small amounts of inorganic, organic components and solvents residues can be generated during such a process. These components left over in the API are considered to be impurities. Therefore, any irrelevant material present in the drug substance even in small amounts might influence the safety and efficacy of the pharmaceutical products. This material is considered to be an impurity even if it is completely inert or has more pharmacological activity (23–26). Up till now, no reported method has been described yet to analyze both drugs, VAL and SAC, combined as supramolecular complex (LCZ696) in dosage form (Entresto™) with an intermediate product of SAC as related substance (BMP). The aim of the current work is to analyze LCZ696 (supramolecular complex of VAL and SAC) in presence of SAC-related substance at low levels relative to the parent drug using three chromatographic methods: HPLC-DAD, HPLC-FLD and HPTLC. The three methods allow peak purity confirmation. However, the methods show diverse sensitivities which affect the impurity detection and quantitation levels to the parent drug. Therefore, this work deals with the development of an accurate and efficient analytical method to determine the quality of the final product (tablets), which is a crucial activity during the development process of drug product in generic pharmaceutical industries. Experimental Instrumentation The HPLC-DAD system consisted of Agilent 1200 series (auto-injector, quaternary pump, vacuum degasser and diode array and multiple wavelength detectors G1315 C/D and G1365 C/D) and FLD in series connected to a computer loaded with Agilent ChemStation Software (Agilent Technologies, Santa Clara, CA, USA). The FLD was Agilent 1260 Infinity Fluorescence Detector for programmable single wavelength (excitation and emission) detection up to 74 Hz data rate (G1321C). For sonication, JP SELECTA, SA sonicator was used (Abrera, Barcelona, Spain). pH meter was from Crison Instruments, SA (Barcelona). HPTLC plates (20 × 10 cm, aluminum plates with 250-μm thickness precoated with silica gel 60 F254) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100-μL CAMAG Microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Linomat IV Applicator (CAMAG, Muttenz, Switzerland). The slit dimensions were 5.00 × 0.45 mm and the scanning speed was 20 mm s–1. Ascending development of the mobile phase was carried out in a CAMAG 20 × 10 cm twin trough glass chamber. The optimized chamber saturation time for mobile phase was 20 min at room temperature (25 ± 2 C˚) with 20 mL mobile phase volume. Densitometric scanning was performed at 255 nm on a CAMAG TLC Scanner 3 (CAMAG, Muttenz, Switzerland) operated in the reflectance–absorbance mode and controlled by CAMAG CATS Software (V 3.15) (CAMAG, Muttenz, Switzerland). The source of radiation utilized was deuterium lamp emitting a continuous ultraviolet spectrum between 190 and 400 nm. Materials VAL (99%) was kindly supplied by Medizen Pharmaceutical Industries Company, Egypt. SAC (99.5%) and SAC-related substance (BMP) were kindly supplied by Abblis Chemical Company, China. The pharmaceutical formulation analyzed was Entresto™ tablets (label claim: 25.7 mg VAL and 24.3 mg SAC per tablet formulated as a salt complex of the anionic forms of SAC and VAL, sodium cations and water molecules in the molar ratio of 1:1:3:2.5, respectively, Leduc Rexall Drug Store, Canada). HPLC-Grade Chloroform (Fisher Scientific, UK), methanol and ethyl acetate (Gliwice, ul. Sowinskiego 11, Poland) and analytical grade of glacial acetic acid and ammonia (EL-Nasr Chemical Co, Egypt) were used in the experiments. Standard solutions For the proposed chromatographic methods, stock solutions containing 1 mg mL−1 of each of VAL, SAC and BMP standard solution prepared in HPLC-grade methanol were stored in refrigerator at 4°C for a week. For HPLC-DAD and HPLC-FLD, working stocks of 100 and 10 μg mL−1 were prepared in HPLC-grade methanol for analyzing each of the three studied compounds, respectively. The final working standard solutions were prepared by diluting aliquots of the working stocks with distilled water to reach the concentration ranges 0.2–20 μg mL−1 for VAL, SAC and 0.05–20 μg mL−1 for BMP upon using DAD. Whereas upon using FLD, concentration ranges of 0.025–10, 0.01–3 and 0.005–1 μg mL−1 for VAL, SAC and BMP, respectively, were reached. For HPTLC, a working stock of 100 μg mL−1 was prepared in HPLC-grade methanol for BMP. The final working standard solutions were prepared by diluting aliquots from VAL or SAC stock solution or BMP working stock solution in 10-mL volumetric flasks with HPLC-grade methanol. This was done in order to reach the concentration ranges 10–100 μg mL−1 for both VAL and SAC; and 2–20 μg mL−1 for BMP. Chromatographic conditions HPLC method A mobile phase system consisting of acetonitrile and 25 mM phosphate buffer of pH 3 (sodium dihydrogen phosphate monohydrate adjusted with orthophosphoric acid) in a ratio 65:35 (v/v) were used. The mobile phase was degassed and filtered by passing through 0.45 μm Millipore filter. All over the run, 0.9 mL min−1 flow rate was maintained. Setting the injection volume at 20 μL was attained. The analytes were monitored by the DAD which was set at 255 nm as it provided high sensitivity for BMP as SAC-related substance, in the same time it gave reasonable responses with VAL and SAC, and by FLD which was set at different λex/λem (nm/nm): 0–4.5 min at 255/374; 4.5–6 min at 255/314, for monitoring and quantitation of both VAL, SAC and BMP. All determinations were performed at ambient temperature. For each concentration, the injections were carried out in triplicate. HPTLC method From each working standard solution, portions equal to 10 μL were spotted on HPTLC in the form of bands. A distance of 10 mm was chosen to separate these bands apart while 20 mm was set to separate them from the plate bottom. Triplicate applications were made for each solution. The plate was then developed using chloroform–ethyl acetate–glacial acetic acid (10:10:0.1, v/v) as a mobile phase. For all the proposed chromatographic methods, the area values of analyte’s peaks were plotted against the corresponding concentrations to attain calibration curves and regression equations, Table I. Table I Regression and statistical parameters for the determination of VAL, SAC and BMP using HPLC-DAD, HPLC-FD and HPTLC methods   VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6    VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6  *Linearity range is in microgram per milliliter. aIntercept. bStandard deviation of the intercept. cSlope. dStandard deviation of the slope. eStandard deviation of residuals. fCorrelation coefficient. gLOD = Limit of detection (μg mL−1). hLOQ = Limit of quantitation (μg mL−1). iVariance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Table I Regression and statistical parameters for the determination of VAL, SAC and BMP using HPLC-DAD, HPLC-FD and HPTLC methods   VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6    VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6  *Linearity range is in microgram per milliliter. aIntercept. bStandard deviation of the intercept. cSlope. dStandard deviation of the slope. eStandard deviation of residuals. fCorrelation coefficient. gLOD = Limit of detection (μg mL−1). hLOQ = Limit of quantitation (μg mL−1). iVariance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Assay of commercial tablets Ten Entresto™ tablets were weighed and finely powdered. HPLC-grade methanol (15 mL) was added to a quantity of the powdered tablets equivalent to one tablet. This solution is sonicated for 20 min. Filtered through Whatman no. 1 filter paper followed by quantitative transfer of the filtered solution into 25-mL volumetric flask and diluted to volume with methanol. For HPTLC method, an aliquot of 0.4 mL was transferred to volumetric flasks (volume of 10 mL) and diluted with the aid of methanol to obtain the target concentration within the linear range of each studied drug. While for HPLC methods, a further dilution was needed to reach final concentration of 10.28 and 9.72 μg mL−1 for VAL and SAC, respectively. Then an aliquot (0.5 mL) of the diluted solution was transferred to 10-mL volumetric flask and diluted with distilled water to obtain concentrations within the linear range of each studied drug. The general procedure described above either for HPLC-DAD, HPLC-FLD or HPTLC was followed. Results Due to the lack of reports analyzing VAL and SAC along with SAC-related substance (BMP) in literature, it was important to establish different chromatographic methods with varied sensitivities to allow their separation and quantitation especially BMP, which is needed to be quantified at low levels in presence of parent drug. That is why methods optimization was directed towards achieving the best separation, highest sensitivity and lowest baseline noise. Moreover, testing the tablets for the presence of BMP was done. Methods development and optimization HPLC-DAD/FLD First trials involve the use of different reversed-phase columns: Zorbax SB-C8 (250 × 4.6 mm, particle size 5 μ), Zorbax Eclipse Plus-C18 (150 × 4.6 mm, particle size 3.5 μ) and Zorbax Eclipse Plus-C18 (250 × 4.6 mm, particle size 5 μ). Discussion of the results to optimize conditions is in section Discussion, Supplementary data Figures S1 and S2. The system suitability parameters were tested for HPLC methods and listed in Table II. According to USP, these parameters were in the accepted ranges. Figure 2 shows the separation of VAL (at 4.2 min), BMP (at 4.6 min) and SAC (at 5.1 min) with suitable sharpness, resolution and peak symmetry by using both DAD and FLD. Table II System suitability parameters for the HPLC-DAD/FD methods for determination of VAL, BMP and SAC Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  System suitability recommendations: k’(1–10), N > 2,000, α > 1, Rs > 2 and Af (0.8–1.2). *Average tR ± SD of three determinations. Table II System suitability parameters for the HPLC-DAD/FD methods for determination of VAL, BMP and SAC Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  System suitability recommendations: k’(1–10), N > 2,000, α > 1, Rs > 2 and Af (0.8–1.2). *Average tR ± SD of three determinations. Figure 2. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.5 μg mL−1 of each of VAL, BMP and SAC (tR = 4.25, 4.60 and 5.15 min, respectively) (a) using DAD, (a’) using FLD and their corresponding purity profiles and plots obtained by DAD for VAL (b,b’), BMP (c,c’) and SAC (d,d’). Figure 2. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.5 μg mL−1 of each of VAL, BMP and SAC (tR = 4.25, 4.60 and 5.15 min, respectively) (a) using DAD, (a’) using FLD and their corresponding purity profiles and plots obtained by DAD for VAL (b,b’), BMP (c,c’) and SAC (d,d’). HPTLC Optimization of HPTLC method parameters is important for the simultaneous determination of the three compounds in their mixtures, in addition to attaining symmetric peak shape and reproducible retardation factor (RF) values. A full study of different experimental conditions such as mobile phase composition and detection wavelength was done to optimize these conditions to provide accurate, precise and reproducible, compact, flat bands for the simultaneous determination of VAL, SAC and BMP. Figure 3 shows that the three compounds in mixtures could be separated with good resolution as sharp and symmetrical peaks with RF 0.25, 0.32, and 0.42 for VAL, SAC and BMP, respectively, upon the use of a mobile phase consisting of chloroform–ethyl acetate–glacial acetic acid. Discussion of the results to optimize conditions is in section Discussion. Figure 3. View largeDownload slide Typical densitogram (a) of 40, 100 and 3 μg mL−1 of VAL (1), SAC (2) and BMP (3), respectively, with their spectra illustrating peak purity of (b) VAL, (c) SAC and (d) BMP; each is obtained from corresponding standards. Figure 3. View largeDownload slide Typical densitogram (a) of 40, 100 and 3 μg mL−1 of VAL (1), SAC (2) and BMP (3), respectively, with their spectra illustrating peak purity of (b) VAL, (c) SAC and (d) BMP; each is obtained from corresponding standards. The optimum system for the adequate separation of the three compounds with reasonable RF values was chloroform: ethyl acetate: glacial acetic acid in a ratio of 10:10:0.1, v/v. For the selection of optimum scanning wavelength, different wavelengths were tried for the simultaneous determination of the ternary mixture. A wavelength of 255 nm was selected as it provided high sensitivity for the determination of BMP as SAC-related substance in the presence of other drugs, at the same time it gave a reasonable response for VAL and SAC. Methods validation International conference on harmonization guidelines were used for validating the different chromatographic methods used for analyzing the two drugs in presence of related substance, BMP (27). Linearity and concentration ranges Series of different concentrations of each compound were analyzed to evaluate the linearity of each proposed chromatographic method. Least squares treatment of the calibration data was done to generate the linear regression equations. Under the optimized conditions for each method, the measured peak areas obtained either upon using HPLC-DAD, HPLC-FLD or HPTLC were found proportional to VAL, SAC and BMP concentrations. Excellent linearity indicated by high r and F values with low Sy/x and significant F values (28,29), Table I. Detection and quantification limits For HPLC and HPTLC methods, both of limits of detection (LOD) and limits of quantitation (LOQ) were determined practically by estimating the analyte concentration having a signal-to-noise ratio 3:1 and 10:1, respectively. These concentrations were verified practically. As can be seen in Table I, the HPLC-FLD yielded low LOD (8.3, 3.3 and 1.7 ng mL−1) and LOQ (25, 10 and 5 ng mL−1) values for VAL, SAC and BMP, respectively. Accuracy and precision Testing the proposed methods for their satisfactory ability of quantifying the compounds under study with adequate accuracy and precision was done over a wide range of ratios (Supplementary data Tables S3 and S4). Both of the recovery percent and relative error (Er %) were calculated so as to test the method accuracy (27,29). The excellent values of recoveries (with value not <98% and not >102%) achieved in company with low percentage error (values recorded are <2%) point out that the proposed methods were of high accuracy. The percentage relative standard deviation (RSD%) values which were achieved after conducting repeatability and intermediate precision were <2% pointing out the good compliance among the individual test results thus proving that the proposed methods were of high precision, (Supplementary data Tables S3 and S4). Moreover, the proposed methods could analyze accurately and precisely SAC in presence of its related substance BMP as impurity at 1% level (SAC:BMP, 20:0.2) upon using HPLC-DAD whereas at 0.167% upon using HPLC-FLD (SAC:BMP, 3:0.005). While upon using HPTLC method, SAC in presence of its related substance BMP as impurity could be accurately and precisely analyzed at 3% level. Specificity The specificity was determined by the absence of any interference from the normally existing excipients and additives in dosage form with the applied methods as will be revealed later in assay section of tablets dosage form. Both VAL and SAC peaks were well separated from each other and from SAC-related substance ‘BMP’. This was confirmed as resolution values of 2.38 and 3.12 were obtained upon using HPLC-DAD whereas 1.85 and 2.47 upon using HPLC-FLD for VAL, BMP and BMP, SAC, respectively. Also for HPTLC, complete separation of VAL and SAC peaks from each other and from SAC-related substance ‘impurity’ was attained. Spiking procedures were done to confirm the absence of BMP in SAC pure substance and LCZ696 complex in tablet. For analysis in dosage form, no signs of BMP peak in dosage form or foreign peaks due to excipients were found (Figure 4). Moreover, the purity profiles obtained by DAD for HPLC method indicate the purity of SAC and VAL peaks in dosage form analysis, Figure 4. Also, peak purity profiling performed by the WinCATS software for HPTLC ascertains the purity of the peaks. As seen in Figure 5, spots were affirmed pure as the extracted spectra at various points were laid over one another and the values which were calculated as correlation coefficients were acceptable (>0.999). Figure 4. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.514 μg mL−1 VAL and 0.486 μg mL−1 SAC obtained from tablet (dosage form) using DAD (a) and FLD (a’), and their corresponding purity profiles and plots of VAL (b, b’) and SAC (c, c’) obtained by DAD. Figure 4. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.514 μg mL−1 VAL and 0.486 μg mL−1 SAC obtained from tablet (dosage form) using DAD (a) and FLD (a’), and their corresponding purity profiles and plots of VAL (b, b’) and SAC (c, c’) obtained by DAD. Figure 5. View largeDownload slide A typical HPTLC densitogram (a) of dosage form (Entresto™) 41.12 and 38.88 μg mL−1 with spectra illustrating peak purity of (b) VAL and (c) SAC in dosage form. Figure 5. View largeDownload slide A typical HPTLC densitogram (a) of dosage form (Entresto™) 41.12 and 38.88 μg mL−1 with spectra illustrating peak purity of (b) VAL and (c) SAC in dosage form. Robustness Various chromatographic conditions were slightly altered to validate the method robustness as: source of acetonitrile (Gliwice, ul. Sowinskiego 11, Poland-T26 Stillorgan Ind. Park, Co, Dublin, Ireland), working wavelength (±1 nm), ratio of mobile phase (±2%), pH of buffer (±0.2 pH units) and column temperature (±1°C) for HPLC-DAD. While for HPLC-FLD, source of acetonitrile, pH of buffer (±0.2 pH units), column temperature (±1˚C) and excitation and emission wavelength (±1 nm) were varied. For HPTLC robustness appraisal, minor variations in the adjusted method parameters were done. The solvents ratio of the mobile phase and the time for chamber saturation were subjected to variation in the range of ±2 ml and ±10 min, respectively, of the used optimized conditions. The amount of mobile phase was varied by ±5 ml, and changing the detection wavelength was also varied by ±1 nm. The effect of these changes on the RF values and peak area were studied. The percentage standard deviation of the peak areas for each compound was calculated. The low values obtained for RSD% in company with almost unaltered RF and retention times values obtained after introducing small deliberate changes in the methods parameters indicated the robustness of the developed methods (Supplementary data Tables S5−S7). Stability of solutions For VAL, SAC and BMP final working standard solutions, no chromatographic changes were observed within 6 h at room temperature. Also, the stock and working stock solutions of the analytes were found to be stable for at least one week when kept in refrigerator at 4°C. No significant degradation was detected during this time interval besides RF and retention times values and peak areas of the drugs remained unchanged. Assay of tablets dosage form The developed chromatographic methods were able to determine VAL and SAC supramolecular complex ‘LCZ696’ in dosage form (Entresto™). The drugs were eluted at their specific RF and retention time values with no interfering peaks were observed from any of the inactive ingredients (Figures 4 and 5). The assay results revealed that satisfactory results were obtained for accuracy and precision as indicated from recovery %, SD, RSD% and Er (%) values (Table III). With the aim of comparing the results statistically, the results of the proposed methods were compared with the results of HPLC report (18). The one-way analysis of variance test (Single factor ANOVA) was carried out. The ANOVA test is a valuable statistical tool for comparing recovery data obtained from more than two methods for analysis (29). The calculated F (n = 6) values 2.52 and 2.34 for VAL and SAC assay, respectively, did not exceed the critical value (3.10), indicating that absence of major differences between the proposed methods at P = 0.05. Table III Assay results for the determination of VAL and SAC in dosage form using the proposed HPLC-DAD, HPLC-FD and HPTLC methods (n = 6) Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  *Concentration is in microgram per milliliter. aMean recovery of the found concentration ± standard deviation for six determinations. b% Relative standard deviation. c% Relative error. Table III Assay results for the determination of VAL and SAC in dosage form using the proposed HPLC-DAD, HPLC-FD and HPTLC methods (n = 6) Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  *Concentration is in microgram per milliliter. aMean recovery of the found concentration ± standard deviation for six determinations. b% Relative standard deviation. c% Relative error. Discussion For HPLC methods, Long C18 Column gave the best compromise regarding peak separation, symmetry peaks and resolution with quite suitable retention time. Therefore, it was used for the analysis. On the other hand, other columns gave broad VAL peak and it was tailed or shouldered with poor separation from SAC. For reaching the optimum acetonitrile ratio in mobile phase, different ratios were tried to attain a suitable separation of the three compounds with reasonable retention, Supplementary data Figures S1a–e. Acetonitrile with a ratio of 50% in the mobile phase caused the three compounds to elute very late (retention time >7 min). However, 70% acetonitrile causes rapid elution of the compounds with poor resolution (Rs <1.7), Supplementary data Figure S2. In addition, 55% and 60% acetonitrile yielded overlapped peaks. It was found that 65% acetonitrile in the mobile phase provided a good separation of the related substance peak (BMP) from the parent drugs with reasonable retention time for the three compounds. For adjusting the pH of phosphate buffer (0.025 M sodium dihydrogen phosphate), different pH values of the phosphate buffer were investigated in its ratio with acetonitrile (35:65 v/v), taking into consideration the pKa values of the compounds. It was found that pH 3 is the optimum as pH value ≥7 caused splitting of BMP peak and broadening of VAL peak. The ionic strength of the buffer had no effect on the peaks elution or shape. Thus, 25 mM was used during the study. With the aim of finding the best wavelength in order to quantify both VAL and SAC in the presence of SAC-related substance, several wavelengths were tried for both DAD and FLD. This was done in order to find the optimum wavelength which gives a maximum response for BMP and reasonable responses for our targeted drugs. So, the DAD was set at 255 nm Figure 2. Similarly, different excitation and emission wavelengths were tried for FLD. After careful study, the FLD was set at different λex/λem (nm/nm) in a program mode. First, FLD was set at 255/374 (λex/λem) to analyze VAL with good sensitivity. After that, for analyzing SAC-related substance (BMP) in presence of its parent drug, SAC, FLD was set at 255/314 (λex/λem). This provides the analysis of BMP at low levels in presence of SAC. For HPTLC method, several trials were done to select a solvent system that would give dense and compact spots with significantly different RF values for the three compounds. According to HPTLC reports found in literature for VAL analysis, different systems containing mixture of solvents were tried with different ratios: (chloroform:methanol:acetic acid), (chloroform, ethyl acetate, methanol:acetic acid), (chloroform, toluene, methanol:acetic acid), (toluene, methanol, ethyl acetate:acetic acid). All these systems yielded poorly separated and overlapped spots. The system of chloroform:ethyl acetate:acetic acid was the most appropriate mobile phase. Different ratios of chloroform and ethyl acetate were tried 1:1, 2:1 and 1:2. Equal ratio of them provides the optimum separation with reasonable RF values for the three compounds. Upon using lower percentage of chloroform (low polarity solvent) than the optimum one, lower RF values were obtained for VAL beside the bad resolution between the analytes. Also, it was found that upon using the mobile phase system of chloroform:ethyl acetate in 1:1 ratio without acetic acid (HAC), distorted bands with low RF values and also poor resolution between VAL and BMP were obtained. Also, upon using ammonia (33%) instead of HAC, the three compounds were retained on the starting line. For the validation of the suggested methods, it can be indicating from the slope values that the HPLC-FLD enables sensitive detection of trace-level analytes and impurities when compared to the HPLC-DAD or HPTLC. In the same way, the LOD and LOQ values are indicative to the low noise levels obtained by the proposed method with high response of the analyte upon using FLD. For tablet dosage form analysis, no sign of BMP as related substance in tablets was noticed. This was ascertained by the absence of any peak at BMP specific RF and retention time. Confirming the absence of SAC-related substance, (BMP) from Entresto™ tablets was done by spiking procedures. Although the proposed HPLC-FLD could quantify BMP at a level of 0.167% of the parent drug and could detect it at a concentration of 5 ng ml−1 but no sign of BMP was detected in tablets. Moreover, peak purity was confirmed through the peak purity profiling (Figure 4). This was accomplished by putting side by side the recorded spectra at various points of the chromatographic peak; the calculation of a correlation coefficient which exceeds 0.999 was an indication of the peaks homogeneity. Moreover, the HPTLC Wincats software could test the purity of dosage form sample peaks by comparing their spectra with those of the standard peaks. The complete superimpose of these recorded spectra confirms the purity of dosage from sample peaks (Figure 5). Conclusion Going through the literature revealed that there were no reports for the analysis and separation of the LCZ696 supramolecular complex components, VAL and SAC, along with SAC-related substance, BMP. The work deals with the development of accurate and efficient different chromatographic techniques, HPLC-DAD/FLD and HPTLC, to determine the quality of the final product (LCZ696 tablets) which is a crucial activity during the development process of this newly approved drug product. The methods show diverse sensitivities which affect the impurity detection and quantitation levels to the parent drug. The HPLC-FLD could successfully analyze BMP at a level of 0.167% of its parent drug and it yielded high slope values indicating its high sensitivity for BMP determination. This indicates that HPLC-FLD was the method of choice for impurity detection, quantitation and monitoring. Supplementary Data Supplementary materials are available at Journal of Chromatographic Science online. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References 1 Segura, J., Salazar, J., Ruilope, L.M.; Dual neurohormonal intervention in CV disease: angiotensin receptor and Neprilysin inhibition; Expert Opin Investig Drugs , ( 2013); 22( 7): 915– 925. 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(accessed 15 March, 2018) 28 Armitage, P., Berry, G.G., Matthews, J.N.S.; Statistical methods in medical research , 3rd ed. Blackwell, Oxford, Boston England, ( 1994). 29 Miller, J.N., Miller, J.C.; Statistics and chemometrics for analytical chemistry: Pearson . Prentice Hall, Harlow, England; Newyork, ( 2005). © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

High performance thin-layer and high performance liquid chromatography coupled with photodiode array and fluorescence detectors for analysis of valsartan and sacubitril in their supramolecular complex with quantitation of sacubitril-related substance in raw material and tablets

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Abstract

Abstract Valsartan (VAL) and sacubitril (SAC) are combined in a supramolecular complex, LCZ696, which is a newly approved remedy for heart failure. SAC-related substance (biphenyl methyl pyrrolidinone [BMP]) which also appears as an intermediate during SAC synthesis is considered to be a suspected impurity for SAC and/or LCZ696 tablets. The study investigates the analysis of VAL and SAC in their supramolecular complex along with SAC-related substance, BMP, using high performance thin-layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) with two different detectors; fluorescence detector (FLD) and diode array detector (DAD). The work aimed at analyzing BMP at low levels in the presence of its parent drug, SAC. BMP was successfully analyzed at a level of 0.167, 1 and 3% of its parent drug, SAC upon using HPLC-FLD, HPLC-DAD and HPTLC, respectively. For HPLC-FLD, the detector was set at λex/λem (nm/nm): 0–4.5 min at 255/374; 4.5–6 min at 255/314, for achieving an adequate sensitivity of the method to monitor and quantify VAL and SAC in the presence of BMP. Low limits of detection (8.3, 3.3 and 1.7 ng mL−1) and limits of quantitation (25, 10 and 5 ng mL−1) values obtained for VAL, SAC and BMP, respectively, upon using FLD suggest that low level of baseline noise enables the detection and quantitation of low BMP concentration. Introduction The new remedy of LCZ696 supramolecular complex is claimed to assist reducing the possibility of cardiovascular mortality and heart failure hospitalization in patients (1). Moreover, it is recommended to be used instead of angiotensin converting enzyme inhibitors or angiotensin II receptor blocker for first-line treatment of these patients (2–4). Valsartan (VAL) is N-pentanoyl-N-{[2′-(1H-tetrazol-5-yl) [1,1′-biphenyl]−4-yl] methyl}-l-valine (Figure 1a). It is mostly used for treatment of high blood pressure and congestive heart failure (5). Different chromatographic methods were found in literature assaying VAL either alone or in combination with other antihypertensive medications including high performance liquid chromatography fluorescence detector (HPLC-FLD) (6,7), high performance thin-layer chromatography (HPTLC) (8–13) and HPLC-diode array detector (HPLC-DAD) (8,14,15). Figure 1. View largeDownload slide Structures of valsartan (a), sacubitril (b), intermediate (c) and their supramolecular complex, LCZ696 (d). Figure 1. View largeDownload slide Structures of valsartan (a), sacubitril (b), intermediate (c) and their supramolecular complex, LCZ696 (d). Sacubitril (SAC) is 4-{[(1 S,3 R)-1-([1,1′-biphenyl]-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl] amino}-4-oxobutanoic acid (Figure 1b). It is a pro-drug, its active metabolite prevents neprilysin which is a neutral endopeptidase (16). So far nothing has been found in literature regarding this novel combination LCZ696 (VAL/SAC) except four works of literature. One of them is reversed phase-HPLC method for their assaying in tablets, another one is LC-MS/MS which deals with their quantification in rat plasma while the most recent two reports are the authors’ works, the first is investigating the spectrofluorimetric behavior of SAC and analyzing it with VAL in LCZ696 using derivative-based spectrofluorimetric method while the second investigates the SAC degradation kinetics with its HPLC analysis in LCZ696. (17–20). SAC-related substance (biphenyl methyl pyrrolidinone [BMP]) is (S)-5-biphenyl-4-ylmethylpyrrolidin-2-one (Figure 1c). It is found in the pathway of synthesis of SAC as intermediate (21,22). So, it is considered to be a suspected impurity that could be found either in SAC raw materials and/or LCZ696 (Figure 1d) tablets. Most active pharmaceutical ingredients (API) are being synthesized by organic chemical syntheses. Various components, for instance: very small amounts of inorganic, organic components and solvents residues can be generated during such a process. These components left over in the API are considered to be impurities. Therefore, any irrelevant material present in the drug substance even in small amounts might influence the safety and efficacy of the pharmaceutical products. This material is considered to be an impurity even if it is completely inert or has more pharmacological activity (23–26). Up till now, no reported method has been described yet to analyze both drugs, VAL and SAC, combined as supramolecular complex (LCZ696) in dosage form (Entresto™) with an intermediate product of SAC as related substance (BMP). The aim of the current work is to analyze LCZ696 (supramolecular complex of VAL and SAC) in presence of SAC-related substance at low levels relative to the parent drug using three chromatographic methods: HPLC-DAD, HPLC-FLD and HPTLC. The three methods allow peak purity confirmation. However, the methods show diverse sensitivities which affect the impurity detection and quantitation levels to the parent drug. Therefore, this work deals with the development of an accurate and efficient analytical method to determine the quality of the final product (tablets), which is a crucial activity during the development process of drug product in generic pharmaceutical industries. Experimental Instrumentation The HPLC-DAD system consisted of Agilent 1200 series (auto-injector, quaternary pump, vacuum degasser and diode array and multiple wavelength detectors G1315 C/D and G1365 C/D) and FLD in series connected to a computer loaded with Agilent ChemStation Software (Agilent Technologies, Santa Clara, CA, USA). The FLD was Agilent 1260 Infinity Fluorescence Detector for programmable single wavelength (excitation and emission) detection up to 74 Hz data rate (G1321C). For sonication, JP SELECTA, SA sonicator was used (Abrera, Barcelona, Spain). pH meter was from Crison Instruments, SA (Barcelona). HPTLC plates (20 × 10 cm, aluminum plates with 250-μm thickness precoated with silica gel 60 F254) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100-μL CAMAG Microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Linomat IV Applicator (CAMAG, Muttenz, Switzerland). The slit dimensions were 5.00 × 0.45 mm and the scanning speed was 20 mm s–1. Ascending development of the mobile phase was carried out in a CAMAG 20 × 10 cm twin trough glass chamber. The optimized chamber saturation time for mobile phase was 20 min at room temperature (25 ± 2 C˚) with 20 mL mobile phase volume. Densitometric scanning was performed at 255 nm on a CAMAG TLC Scanner 3 (CAMAG, Muttenz, Switzerland) operated in the reflectance–absorbance mode and controlled by CAMAG CATS Software (V 3.15) (CAMAG, Muttenz, Switzerland). The source of radiation utilized was deuterium lamp emitting a continuous ultraviolet spectrum between 190 and 400 nm. Materials VAL (99%) was kindly supplied by Medizen Pharmaceutical Industries Company, Egypt. SAC (99.5%) and SAC-related substance (BMP) were kindly supplied by Abblis Chemical Company, China. The pharmaceutical formulation analyzed was Entresto™ tablets (label claim: 25.7 mg VAL and 24.3 mg SAC per tablet formulated as a salt complex of the anionic forms of SAC and VAL, sodium cations and water molecules in the molar ratio of 1:1:3:2.5, respectively, Leduc Rexall Drug Store, Canada). HPLC-Grade Chloroform (Fisher Scientific, UK), methanol and ethyl acetate (Gliwice, ul. Sowinskiego 11, Poland) and analytical grade of glacial acetic acid and ammonia (EL-Nasr Chemical Co, Egypt) were used in the experiments. Standard solutions For the proposed chromatographic methods, stock solutions containing 1 mg mL−1 of each of VAL, SAC and BMP standard solution prepared in HPLC-grade methanol were stored in refrigerator at 4°C for a week. For HPLC-DAD and HPLC-FLD, working stocks of 100 and 10 μg mL−1 were prepared in HPLC-grade methanol for analyzing each of the three studied compounds, respectively. The final working standard solutions were prepared by diluting aliquots of the working stocks with distilled water to reach the concentration ranges 0.2–20 μg mL−1 for VAL, SAC and 0.05–20 μg mL−1 for BMP upon using DAD. Whereas upon using FLD, concentration ranges of 0.025–10, 0.01–3 and 0.005–1 μg mL−1 for VAL, SAC and BMP, respectively, were reached. For HPTLC, a working stock of 100 μg mL−1 was prepared in HPLC-grade methanol for BMP. The final working standard solutions were prepared by diluting aliquots from VAL or SAC stock solution or BMP working stock solution in 10-mL volumetric flasks with HPLC-grade methanol. This was done in order to reach the concentration ranges 10–100 μg mL−1 for both VAL and SAC; and 2–20 μg mL−1 for BMP. Chromatographic conditions HPLC method A mobile phase system consisting of acetonitrile and 25 mM phosphate buffer of pH 3 (sodium dihydrogen phosphate monohydrate adjusted with orthophosphoric acid) in a ratio 65:35 (v/v) were used. The mobile phase was degassed and filtered by passing through 0.45 μm Millipore filter. All over the run, 0.9 mL min−1 flow rate was maintained. Setting the injection volume at 20 μL was attained. The analytes were monitored by the DAD which was set at 255 nm as it provided high sensitivity for BMP as SAC-related substance, in the same time it gave reasonable responses with VAL and SAC, and by FLD which was set at different λex/λem (nm/nm): 0–4.5 min at 255/374; 4.5–6 min at 255/314, for monitoring and quantitation of both VAL, SAC and BMP. All determinations were performed at ambient temperature. For each concentration, the injections were carried out in triplicate. HPTLC method From each working standard solution, portions equal to 10 μL were spotted on HPTLC in the form of bands. A distance of 10 mm was chosen to separate these bands apart while 20 mm was set to separate them from the plate bottom. Triplicate applications were made for each solution. The plate was then developed using chloroform–ethyl acetate–glacial acetic acid (10:10:0.1, v/v) as a mobile phase. For all the proposed chromatographic methods, the area values of analyte’s peaks were plotted against the corresponding concentrations to attain calibration curves and regression equations, Table I. Table I Regression and statistical parameters for the determination of VAL, SAC and BMP using HPLC-DAD, HPLC-FD and HPTLC methods   VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6    VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6  *Linearity range is in microgram per milliliter. aIntercept. bStandard deviation of the intercept. cSlope. dStandard deviation of the slope. eStandard deviation of residuals. fCorrelation coefficient. gLOD = Limit of detection (μg mL−1). hLOQ = Limit of quantitation (μg mL−1). iVariance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Table I Regression and statistical parameters for the determination of VAL, SAC and BMP using HPLC-DAD, HPLC-FD and HPTLC methods   VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6    VAL  SAC  BMP  Parameters  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  HPLC-DAD  HPLC-FD  HPTLC  Linearity range*  0.2–20  0.025–10  10–100  0.2–20  0.01–3  10–100  0.05–20  0.005–1  2–20  aa  −0.05  10.86  −91.18  2.89  15.91  10.62  −0.68  22.44  −90.51  Sab  0.79  4.41  26.46  0.89  5.66  30.23  2.99  4.95  22.64  bc  35.80  324.73  32.88  60.52  1,312.57  36.60  120.59  2,464.05  154.47  Sbd  0.10  1.17  0.41  0.11  5.13  0.47  0.41  12.28  1.97  Sy/xe  1.75  11.27  31.80  1.97  13.92  36.33  7.86  11.36  31.58  rf  0.9999  0.9998  0.9994  0.9999  0.9998  0.9993  0.9998  0.9998  0.9994  LODg  0.067  0.0083  2.87  0.05  0.0033  2.27  0.017  0.0017  0.58  LOQh  0.20  0.025  8.22  0.15  0.01  7.56  0.05  0.005  1.93  Fi  1,39,079  76,612  6,327.19  3,12,658  65,407  6,009.16  87,169  40,293  6,173.57  Significance F  2.50 × 10−14  2.12 × 10−15  4.38 × 10−6  2.21 × 10−15  3.69 × 10−15  4.73 × 10−6  1.94 × 10−17  1.03 × 10−12  4.54 × 10−6  *Linearity range is in microgram per milliliter. aIntercept. bStandard deviation of the intercept. cSlope. dStandard deviation of the slope. eStandard deviation of residuals. fCorrelation coefficient. gLOD = Limit of detection (μg mL−1). hLOQ = Limit of quantitation (μg mL−1). iVariance ratio, equals the mean of squares due to regression divided by the mean of squares about regression (due to residuals). Assay of commercial tablets Ten Entresto™ tablets were weighed and finely powdered. HPLC-grade methanol (15 mL) was added to a quantity of the powdered tablets equivalent to one tablet. This solution is sonicated for 20 min. Filtered through Whatman no. 1 filter paper followed by quantitative transfer of the filtered solution into 25-mL volumetric flask and diluted to volume with methanol. For HPTLC method, an aliquot of 0.4 mL was transferred to volumetric flasks (volume of 10 mL) and diluted with the aid of methanol to obtain the target concentration within the linear range of each studied drug. While for HPLC methods, a further dilution was needed to reach final concentration of 10.28 and 9.72 μg mL−1 for VAL and SAC, respectively. Then an aliquot (0.5 mL) of the diluted solution was transferred to 10-mL volumetric flask and diluted with distilled water to obtain concentrations within the linear range of each studied drug. The general procedure described above either for HPLC-DAD, HPLC-FLD or HPTLC was followed. Results Due to the lack of reports analyzing VAL and SAC along with SAC-related substance (BMP) in literature, it was important to establish different chromatographic methods with varied sensitivities to allow their separation and quantitation especially BMP, which is needed to be quantified at low levels in presence of parent drug. That is why methods optimization was directed towards achieving the best separation, highest sensitivity and lowest baseline noise. Moreover, testing the tablets for the presence of BMP was done. Methods development and optimization HPLC-DAD/FLD First trials involve the use of different reversed-phase columns: Zorbax SB-C8 (250 × 4.6 mm, particle size 5 μ), Zorbax Eclipse Plus-C18 (150 × 4.6 mm, particle size 3.5 μ) and Zorbax Eclipse Plus-C18 (250 × 4.6 mm, particle size 5 μ). Discussion of the results to optimize conditions is in section Discussion, Supplementary data Figures S1 and S2. The system suitability parameters were tested for HPLC methods and listed in Table II. According to USP, these parameters were in the accepted ranges. Figure 2 shows the separation of VAL (at 4.2 min), BMP (at 4.6 min) and SAC (at 5.1 min) with suitable sharpness, resolution and peak symmetry by using both DAD and FLD. Table II System suitability parameters for the HPLC-DAD/FD methods for determination of VAL, BMP and SAC Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  System suitability recommendations: k’(1–10), N > 2,000, α > 1, Rs > 2 and Af (0.8–1.2). *Average tR ± SD of three determinations. Table II System suitability parameters for the HPLC-DAD/FD methods for determination of VAL, BMP and SAC Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  Parameters  VAL  BMP  SAC    HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  HPLC-DAD  HPLC-FD  tR ± SD (min)*  4.2  4.6  5.1  Capacity factor (k’)  1  1.1  1.3  Theoretical plates (N)  10,639  7,350  12,258  7,724  11,918  8,741  Selectivity (α)  1.1    1.18  Resolution (Rs)  2.38  1.85    3.12  2.47  Asymmetry factor (Af)  0.80  0.82  0.80  0.85  0.80  0.82  System suitability recommendations: k’(1–10), N > 2,000, α > 1, Rs > 2 and Af (0.8–1.2). *Average tR ± SD of three determinations. Figure 2. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.5 μg mL−1 of each of VAL, BMP and SAC (tR = 4.25, 4.60 and 5.15 min, respectively) (a) using DAD, (a’) using FLD and their corresponding purity profiles and plots obtained by DAD for VAL (b,b’), BMP (c,c’) and SAC (d,d’). Figure 2. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.5 μg mL−1 of each of VAL, BMP and SAC (tR = 4.25, 4.60 and 5.15 min, respectively) (a) using DAD, (a’) using FLD and their corresponding purity profiles and plots obtained by DAD for VAL (b,b’), BMP (c,c’) and SAC (d,d’). HPTLC Optimization of HPTLC method parameters is important for the simultaneous determination of the three compounds in their mixtures, in addition to attaining symmetric peak shape and reproducible retardation factor (RF) values. A full study of different experimental conditions such as mobile phase composition and detection wavelength was done to optimize these conditions to provide accurate, precise and reproducible, compact, flat bands for the simultaneous determination of VAL, SAC and BMP. Figure 3 shows that the three compounds in mixtures could be separated with good resolution as sharp and symmetrical peaks with RF 0.25, 0.32, and 0.42 for VAL, SAC and BMP, respectively, upon the use of a mobile phase consisting of chloroform–ethyl acetate–glacial acetic acid. Discussion of the results to optimize conditions is in section Discussion. Figure 3. View largeDownload slide Typical densitogram (a) of 40, 100 and 3 μg mL−1 of VAL (1), SAC (2) and BMP (3), respectively, with their spectra illustrating peak purity of (b) VAL, (c) SAC and (d) BMP; each is obtained from corresponding standards. Figure 3. View largeDownload slide Typical densitogram (a) of 40, 100 and 3 μg mL−1 of VAL (1), SAC (2) and BMP (3), respectively, with their spectra illustrating peak purity of (b) VAL, (c) SAC and (d) BMP; each is obtained from corresponding standards. The optimum system for the adequate separation of the three compounds with reasonable RF values was chloroform: ethyl acetate: glacial acetic acid in a ratio of 10:10:0.1, v/v. For the selection of optimum scanning wavelength, different wavelengths were tried for the simultaneous determination of the ternary mixture. A wavelength of 255 nm was selected as it provided high sensitivity for the determination of BMP as SAC-related substance in the presence of other drugs, at the same time it gave a reasonable response for VAL and SAC. Methods validation International conference on harmonization guidelines were used for validating the different chromatographic methods used for analyzing the two drugs in presence of related substance, BMP (27). Linearity and concentration ranges Series of different concentrations of each compound were analyzed to evaluate the linearity of each proposed chromatographic method. Least squares treatment of the calibration data was done to generate the linear regression equations. Under the optimized conditions for each method, the measured peak areas obtained either upon using HPLC-DAD, HPLC-FLD or HPTLC were found proportional to VAL, SAC and BMP concentrations. Excellent linearity indicated by high r and F values with low Sy/x and significant F values (28,29), Table I. Detection and quantification limits For HPLC and HPTLC methods, both of limits of detection (LOD) and limits of quantitation (LOQ) were determined practically by estimating the analyte concentration having a signal-to-noise ratio 3:1 and 10:1, respectively. These concentrations were verified practically. As can be seen in Table I, the HPLC-FLD yielded low LOD (8.3, 3.3 and 1.7 ng mL−1) and LOQ (25, 10 and 5 ng mL−1) values for VAL, SAC and BMP, respectively. Accuracy and precision Testing the proposed methods for their satisfactory ability of quantifying the compounds under study with adequate accuracy and precision was done over a wide range of ratios (Supplementary data Tables S3 and S4). Both of the recovery percent and relative error (Er %) were calculated so as to test the method accuracy (27,29). The excellent values of recoveries (with value not <98% and not >102%) achieved in company with low percentage error (values recorded are <2%) point out that the proposed methods were of high accuracy. The percentage relative standard deviation (RSD%) values which were achieved after conducting repeatability and intermediate precision were <2% pointing out the good compliance among the individual test results thus proving that the proposed methods were of high precision, (Supplementary data Tables S3 and S4). Moreover, the proposed methods could analyze accurately and precisely SAC in presence of its related substance BMP as impurity at 1% level (SAC:BMP, 20:0.2) upon using HPLC-DAD whereas at 0.167% upon using HPLC-FLD (SAC:BMP, 3:0.005). While upon using HPTLC method, SAC in presence of its related substance BMP as impurity could be accurately and precisely analyzed at 3% level. Specificity The specificity was determined by the absence of any interference from the normally existing excipients and additives in dosage form with the applied methods as will be revealed later in assay section of tablets dosage form. Both VAL and SAC peaks were well separated from each other and from SAC-related substance ‘BMP’. This was confirmed as resolution values of 2.38 and 3.12 were obtained upon using HPLC-DAD whereas 1.85 and 2.47 upon using HPLC-FLD for VAL, BMP and BMP, SAC, respectively. Also for HPTLC, complete separation of VAL and SAC peaks from each other and from SAC-related substance ‘impurity’ was attained. Spiking procedures were done to confirm the absence of BMP in SAC pure substance and LCZ696 complex in tablet. For analysis in dosage form, no signs of BMP peak in dosage form or foreign peaks due to excipients were found (Figure 4). Moreover, the purity profiles obtained by DAD for HPLC method indicate the purity of SAC and VAL peaks in dosage form analysis, Figure 4. Also, peak purity profiling performed by the WinCATS software for HPTLC ascertains the purity of the peaks. As seen in Figure 5, spots were affirmed pure as the extracted spectra at various points were laid over one another and the values which were calculated as correlation coefficients were acceptable (>0.999). Figure 4. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.514 μg mL−1 VAL and 0.486 μg mL−1 SAC obtained from tablet (dosage form) using DAD (a) and FLD (a’), and their corresponding purity profiles and plots of VAL (b, b’) and SAC (c, c’) obtained by DAD. Figure 4. View largeDownload slide Typical HPLC chromatograms of a solution containing 0.514 μg mL−1 VAL and 0.486 μg mL−1 SAC obtained from tablet (dosage form) using DAD (a) and FLD (a’), and their corresponding purity profiles and plots of VAL (b, b’) and SAC (c, c’) obtained by DAD. Figure 5. View largeDownload slide A typical HPTLC densitogram (a) of dosage form (Entresto™) 41.12 and 38.88 μg mL−1 with spectra illustrating peak purity of (b) VAL and (c) SAC in dosage form. Figure 5. View largeDownload slide A typical HPTLC densitogram (a) of dosage form (Entresto™) 41.12 and 38.88 μg mL−1 with spectra illustrating peak purity of (b) VAL and (c) SAC in dosage form. Robustness Various chromatographic conditions were slightly altered to validate the method robustness as: source of acetonitrile (Gliwice, ul. Sowinskiego 11, Poland-T26 Stillorgan Ind. Park, Co, Dublin, Ireland), working wavelength (±1 nm), ratio of mobile phase (±2%), pH of buffer (±0.2 pH units) and column temperature (±1°C) for HPLC-DAD. While for HPLC-FLD, source of acetonitrile, pH of buffer (±0.2 pH units), column temperature (±1˚C) and excitation and emission wavelength (±1 nm) were varied. For HPTLC robustness appraisal, minor variations in the adjusted method parameters were done. The solvents ratio of the mobile phase and the time for chamber saturation were subjected to variation in the range of ±2 ml and ±10 min, respectively, of the used optimized conditions. The amount of mobile phase was varied by ±5 ml, and changing the detection wavelength was also varied by ±1 nm. The effect of these changes on the RF values and peak area were studied. The percentage standard deviation of the peak areas for each compound was calculated. The low values obtained for RSD% in company with almost unaltered RF and retention times values obtained after introducing small deliberate changes in the methods parameters indicated the robustness of the developed methods (Supplementary data Tables S5−S7). Stability of solutions For VAL, SAC and BMP final working standard solutions, no chromatographic changes were observed within 6 h at room temperature. Also, the stock and working stock solutions of the analytes were found to be stable for at least one week when kept in refrigerator at 4°C. No significant degradation was detected during this time interval besides RF and retention times values and peak areas of the drugs remained unchanged. Assay of tablets dosage form The developed chromatographic methods were able to determine VAL and SAC supramolecular complex ‘LCZ696’ in dosage form (Entresto™). The drugs were eluted at their specific RF and retention time values with no interfering peaks were observed from any of the inactive ingredients (Figures 4 and 5). The assay results revealed that satisfactory results were obtained for accuracy and precision as indicated from recovery %, SD, RSD% and Er (%) values (Table III). With the aim of comparing the results statistically, the results of the proposed methods were compared with the results of HPLC report (18). The one-way analysis of variance test (Single factor ANOVA) was carried out. The ANOVA test is a valuable statistical tool for comparing recovery data obtained from more than two methods for analysis (29). The calculated F (n = 6) values 2.52 and 2.34 for VAL and SAC assay, respectively, did not exceed the critical value (3.10), indicating that absence of major differences between the proposed methods at P = 0.05. Table III Assay results for the determination of VAL and SAC in dosage form using the proposed HPLC-DAD, HPLC-FD and HPTLC methods (n = 6) Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  *Concentration is in microgram per milliliter. aMean recovery of the found concentration ± standard deviation for six determinations. b% Relative standard deviation. c% Relative error. Table III Assay results for the determination of VAL and SAC in dosage form using the proposed HPLC-DAD, HPLC-FD and HPTLC methods (n = 6) Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  Entresto™ 25.7 mg VAL and 24.3 mg SAC tablets   Concentration*  Mean % recovery ± SDa  RSD (%)b  Er (%)c  VAL  SAC  VAL  SAC  VAL  SAC  VAL  SAC  HPLC-DAD  0.514  0.486  99.17 ± 0.99  100.41 ± 0.88  1.00  0.88  −0.83  0.41  HPLC-FD  0.514  0.486  100.47 ± 1.29  99.73 ± 0.86  1.29  0.87  0.47  −0.27  HPTLC  41.12  38.88  100.69 ± 1.09  100.76 ± 0.82  1.08  0.81  0.69  0.76  *Concentration is in microgram per milliliter. aMean recovery of the found concentration ± standard deviation for six determinations. b% Relative standard deviation. c% Relative error. Discussion For HPLC methods, Long C18 Column gave the best compromise regarding peak separation, symmetry peaks and resolution with quite suitable retention time. Therefore, it was used for the analysis. On the other hand, other columns gave broad VAL peak and it was tailed or shouldered with poor separation from SAC. For reaching the optimum acetonitrile ratio in mobile phase, different ratios were tried to attain a suitable separation of the three compounds with reasonable retention, Supplementary data Figures S1a–e. Acetonitrile with a ratio of 50% in the mobile phase caused the three compounds to elute very late (retention time >7 min). However, 70% acetonitrile causes rapid elution of the compounds with poor resolution (Rs <1.7), Supplementary data Figure S2. In addition, 55% and 60% acetonitrile yielded overlapped peaks. It was found that 65% acetonitrile in the mobile phase provided a good separation of the related substance peak (BMP) from the parent drugs with reasonable retention time for the three compounds. For adjusting the pH of phosphate buffer (0.025 M sodium dihydrogen phosphate), different pH values of the phosphate buffer were investigated in its ratio with acetonitrile (35:65 v/v), taking into consideration the pKa values of the compounds. It was found that pH 3 is the optimum as pH value ≥7 caused splitting of BMP peak and broadening of VAL peak. The ionic strength of the buffer had no effect on the peaks elution or shape. Thus, 25 mM was used during the study. With the aim of finding the best wavelength in order to quantify both VAL and SAC in the presence of SAC-related substance, several wavelengths were tried for both DAD and FLD. This was done in order to find the optimum wavelength which gives a maximum response for BMP and reasonable responses for our targeted drugs. So, the DAD was set at 255 nm Figure 2. Similarly, different excitation and emission wavelengths were tried for FLD. After careful study, the FLD was set at different λex/λem (nm/nm) in a program mode. First, FLD was set at 255/374 (λex/λem) to analyze VAL with good sensitivity. After that, for analyzing SAC-related substance (BMP) in presence of its parent drug, SAC, FLD was set at 255/314 (λex/λem). This provides the analysis of BMP at low levels in presence of SAC. For HPTLC method, several trials were done to select a solvent system that would give dense and compact spots with significantly different RF values for the three compounds. According to HPTLC reports found in literature for VAL analysis, different systems containing mixture of solvents were tried with different ratios: (chloroform:methanol:acetic acid), (chloroform, ethyl acetate, methanol:acetic acid), (chloroform, toluene, methanol:acetic acid), (toluene, methanol, ethyl acetate:acetic acid). All these systems yielded poorly separated and overlapped spots. The system of chloroform:ethyl acetate:acetic acid was the most appropriate mobile phase. Different ratios of chloroform and ethyl acetate were tried 1:1, 2:1 and 1:2. Equal ratio of them provides the optimum separation with reasonable RF values for the three compounds. Upon using lower percentage of chloroform (low polarity solvent) than the optimum one, lower RF values were obtained for VAL beside the bad resolution between the analytes. Also, it was found that upon using the mobile phase system of chloroform:ethyl acetate in 1:1 ratio without acetic acid (HAC), distorted bands with low RF values and also poor resolution between VAL and BMP were obtained. Also, upon using ammonia (33%) instead of HAC, the three compounds were retained on the starting line. For the validation of the suggested methods, it can be indicating from the slope values that the HPLC-FLD enables sensitive detection of trace-level analytes and impurities when compared to the HPLC-DAD or HPTLC. In the same way, the LOD and LOQ values are indicative to the low noise levels obtained by the proposed method with high response of the analyte upon using FLD. For tablet dosage form analysis, no sign of BMP as related substance in tablets was noticed. This was ascertained by the absence of any peak at BMP specific RF and retention time. Confirming the absence of SAC-related substance, (BMP) from Entresto™ tablets was done by spiking procedures. Although the proposed HPLC-FLD could quantify BMP at a level of 0.167% of the parent drug and could detect it at a concentration of 5 ng ml−1 but no sign of BMP was detected in tablets. Moreover, peak purity was confirmed through the peak purity profiling (Figure 4). This was accomplished by putting side by side the recorded spectra at various points of the chromatographic peak; the calculation of a correlation coefficient which exceeds 0.999 was an indication of the peaks homogeneity. Moreover, the HPTLC Wincats software could test the purity of dosage form sample peaks by comparing their spectra with those of the standard peaks. The complete superimpose of these recorded spectra confirms the purity of dosage from sample peaks (Figure 5). Conclusion Going through the literature revealed that there were no reports for the analysis and separation of the LCZ696 supramolecular complex components, VAL and SAC, along with SAC-related substance, BMP. The work deals with the development of accurate and efficient different chromatographic techniques, HPLC-DAD/FLD and HPTLC, to determine the quality of the final product (LCZ696 tablets) which is a crucial activity during the development process of this newly approved drug product. 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Published: Mar 28, 2018

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