Lignin-Based Spherical Structures and Their Use for Improvement of Cilazapril Stability in Solid State.

Biopolymer-based spherical particles exhibit unique properties including narrow sizes and many functional groups on their surfaces. Therefore, they show great potential for application in many scientific and industrial processes. The main aim of this study was to prepare lignin-based spherical particles with the use of a cationic surfactant, hexadecyl(trimethyl)ammonium bromide (CTAB). In the first step, different preparation procedures were tested with varying parameters, including biopolymer and surfactant ratios, lignin filtration, and experimental time. The morphological and dispersion characteristics of the materials were determined to select the best samples with the most promising properties, which could then be tested for their acute toxicity. It was observed that almost all materials were characterized by spherical shapes in micro- and nanosizes. The sample with the best physicochemical properties was used for further analysis and then tested for medical applications: the improvement of the stability of a drug molecule, cilazapril (CIL). The formulated material (CIL@LC-2a 1:1 wt./wt.) exhibited outstanding properties and significantly improved the stability of cilazapril as tested in conditions of increased temperature and humidity. Lignin spherical particles may be employed as a promising material for shielding other active compounds from decomposition.

Multicriteria Optimization Methodology in Stability-Indicating Method Development of Cilazapril and Hydrochlorothiazide.

Multicriteria optimization methodology was applied in development of UHPLC-UV-MS method for separation of cilazapril, hydrochlorothiazide and their degradation products. This method is also applicable for analysis of cilazapril, hydrochlorothiazide and their degradation products in combined tablet formulation. Prior to method optimization forced degradation studies were conducted. Cilazapril and hydrochlorothiazide were subjected to acidic (0.1, 0.5 and 1.0 M HCl), basic (0.1, 0.5 and 1.0 M NaOH), thermal (70°C), oxidative (3-30% H2O2) degradation and photodegradation (day light). Cilazapril appeared to be unstable toward acid and base and resulted in formation of cilazaprilat. Hydrochlorothiazide significantly degraded after acid, base and thermal hydrolysis and formed degradation product was 4-amino-6-chlorobenzene-1.3-disulfonamide. For both substances, after oxidative degradation unknown products have arisen. Initial percentage of acetonitrile in mobile phase, final percentage of acetonitrile in mobile phase, time of gradient elution and column temperature were defined as variables to be optimized toward two chromatographic responses by means of central composite design and Derringer's desirability function. The satisfactory chromatographic analysis was achieved on Kinetex C18 (2.6 µm, 50 × 2.1 mm) column with temperature set at 25°C. The final mobile phase consisted of acetonitrile and 20 mM ammonium formate buffer (pH adjusted to 8.5). The flow rate of the mobile phase was 400 µL min-1 and it was pumped in a gradient elution mode.

Use of LC-MS/TOF, LC-MS(n), NMR and LC-NMR in characterization of stress degradation products: Application to cilazapril.

Forced degradation studies on cilazapril were carried out according to ICH and WHO guidelines. Significant degradation of the drug was observed in acid and base conditions, resulting primarily in cilazaprilat. In neutral condition, five degradation products were formed, while under oxidative condition, two degradation products were generated. In total, seven degradation products were formed, which were separated on an Inertsil C-18 column using a stability-indicating HPLC method. Structure elucidation of the degradation products was done by using sophisticated and hyphenated tools like, LC-MS/TOF, LC-MS(n), on-line H/D exchange, LC-NMR and NMR. Initially, comprehensive mass fragmentation pathway of the drug was laid down. Critical comparison of mass fragmentation pathways of the drug and its hydrolytic degradation products allowed structure characterization of the latter. 1D and 2D proton LC-NMR studies further confirmed the proposed structures of hydrolytic degradation products. The oxidative degradation products could not be characterized using LC-MS and LC-NMR tools. Hence, these degradation products were isolated using preparative HPLC and extensive 1D ((1)H, (13)C, DEPT) and 2D (COSY, TOCSY, HETCOR and HMBC) NMR studies were performed to ascertain their structures. Finally, degradation pathways and mechanisms of degradation of the drug were outlined.

Stability of cilazapril in pediatric oral suspensions prepared from commercially available tablet dosage forms.

Abstract: Cilazapril is a drug commonly used in management of heart failure in pediatric population. On pharmaceutical market it is available only in inconvenient for pediatric use tablet dosage forms. Until now, no oral liquid formulation containing cilazapril has been evaluated. Therefore, the aim of this study was to prepare easy to made and palatable 1 mg/mL oral liquid formulation with cilazapril (with consideration of original and generic cilazapril tablet and different packages) and subsequent investigation of physicochemical stability of these suspensions. Formulations were compounded using cilazapril obtained from original or generic cilazapril marketed tablet formulations and Ora-Blend" suspending agent. Stability of prepared suspensions stored in closed amber glass or amber plastic PET bottles in the temperature of 298 K was estimated throughout 28 day shelf-life period. Chemical stability was assessed by HPLC cilazapril stability indicating method. Physical stability was evaluated by appearance, taste, smell, pH and theological assessments. Cilazapril oral suspensions at concentration of 1 mg/mL demonstrated satisfactory stability over 28 day long storage at room temperature. Cilazapril concentrations remained within acceptable limit (+/- 10%) stored in closed amber bottles made of glass or PET material. Moreover, suspensions physical properties remained unaffected. Cilazapril - Ora-Blend* pediatric oral liquid is easy to made, palatable and stable when stored at room temperature for 28 days. Stability of cilazapril oral liquid remains unchanged while using cilazapril tablets produced by different manufacturers and bottles made of amber glass or PET material.

Cilazapril stability in the presence of hydrochlorothiazide in model mixtures and fixed dose combination.

The presented study aimed at the evaluation of hydrochlorothiazide influence on cilazapril stability in model mixture and fixed dose tablet formulation. The degradation of cilazapril in the presence of hydrochlorothiazide took place according to autocatalytic reaction kinetic mechanism, described mathematically by Prout-Tompkins equation. Hydrochlorothiazide coexistence with cilazapril in model mixture and fixed dose tablet without blister package accelerated cilazapril degradation in comparison with degradation of cilazapril substance. Values of reaction induction time shortened, while those of observed reaction rate constant increased. Increasing values of relative humidity and temperature have negative impact on cilazapril stability. Determined semi-logarithmic relationships: In k = f(RH) and Arrhenius ln k = f(1/T) are linear and are cilazapril stability predictive. The blister (OPA/Alu/PVC//Alu) package of fixed dose tablets, constitutes absolute moisture protection and prevent cilazapril--hydrochlorothiazide interaction occurrence.

Rapid and simple stability indicating HPLC method for the determination of cilazapril in pure substance and pharmaceutical formulation in comparison with classic and derivative spectrophotometric methods.

The present study describes development and subsequent validation of high performance liquid chromatographic method (HPLC) in comparison with spectrophotometric methods (classic, first, second and third order derivative) for determination of pure cilazapril in substance and pharmaceutical preparation. The main aim of this study was to find the method suitable not only for determination of cilazapril, but additionally useful in degradation kinetic study. Only the HPLC method is stability indicating. The HPLC method utilizes LiChroCART 250-4 HPLC-Cartridge, LiChrospher 100 RP-18 (5 µm) column, at ambient temperature, eluted at the flow rate 1.0 mL/min. The mobile phase consists of acetonitrile, methanol and phosphate buffer (pH 2.0) (60:10:30, v/v/v). Wavelength of detection is set at 212 nm. Benzocaine is used as an internal standard. The second and third order derivative spectrophotometric methods can be applied for the cilazapril analysis in substance and tablet, but not for stability evaluation (the lack of selectivity towards degradation product).

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: application to the determination of cilazapril and cilazaprilat in a bioequivalence study.

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril.

Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors.

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.

Spectrophotometric quantitative determination of cilazapril and hydrochlorothiazide in tablets by chemometric methods.

Four chemometric methods were applied to simultaneous determination of cilazapril and hydrochlorothiazide in tablets. Classical least-square (CLS), inverse least-square (ILS), principal component regression (PCR) and partial least-squares (PLS) methods do not need any priori graphical treatment of the overlapping spectra of two drugs in a mixture. For all chemometric calibrations a concentration set of the random mixture consisting of the two drugs in 0.1 M HCI and methanol (1:1) was prepared. The absorbance data in the UV-Vis spectra were measured for the 15 wavelength points (from 222 to 276 nm) in the spectral region 210-290 nm considering the intervals of deltalambda = 4 nm. The calibration of the investigated methods involves only absorbance and concentration data matrices. The developed calibrations were tested for the synthetic mixtures consisting of two drugs and using the Maple V software the chemometric calculations were performed. The results of the methods were compared each other as well as with HPLC method and a good agreement was found.