Investigation of Carcinogenic Impurities of N-Nitrosamines in Sartan Pharmaceutical Products Marketed in Brazil: Development and Validation of Method Based on High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

N-nitrosamines (NA) impurities have unexpectedly been found in sartan products, angiotensin II receptor antagonists that are used to control hypertension, representing an urgent concern for industry, global regulators and for the patients. In this study, an HPLC-MS/MS method was developed and validated for the quantification of six NA (N-nitrosodimethylamine, N-Nitroso-N-methyl-4-aminobutyric acid, N-Nitrosodiethylamine, N-ethyl-N-nitroso-2-propanamine, N-nitroso-diisopropylamine and N-nitroso-di-n-butylamine) in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan products. The method was validated in terms of sensitivity, linearity, accuracy, precision, robustness and stability. The limits of quantification were 100, 31.25, 250, 33, 312.5 and 125 µg kg-1 in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan samples, respectively, which met the sensitivity requirements for the limits set by Food and Drug Administration of the United States. The standard curves showed good linearity. The recoveries ranged from 93.06 to 102.23% in losartan matrix, 83 to 85.9% in valsartan, 96.1 to 101.2% in olmesartan, 89.2 to 97.5% in irbesartan, 93.4 to 132.0% in candesartan and 62.3 to 106.2% in telmisartan matrix. The other parameters met the validation criteria, the good sensitivity and precision, high accuracy and simple and fast analysis provides a reliable method for quality control of NA in sartan pharmaceutical products. The developed method was successfully applied for the determination of N-nitrosamines in 71 sartan products marketed in Brazil.

Genotoxicity evaluation of a valsartan-related complex N-nitroso-impurity.

Recently, the formation of genotoxic and carcinogenic N-nitrosamines impurities during drug manufacturing of tetrazole-containing angiotensin-II blockers has been described. However, drug-related (complex) nitrosamines may also be generated under certain conditions, i.e., through nitrosation of vulnerable amines in drug substances in the presence of nitrite. An investigation of valsartan drug substance showed that a complex API-related N-nitrosamine chemically designated as (S)-2-(((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)(nitroso)amino)-3-methylbutanoic acid (named 181-14) may be generated. 181-14 was shown to be devoid of a mutagenic potential in the Non-GLP Ames test. According to ICH M7 (R1) (2018), impurities that are not mutagenic in the Ames test would be considered Class 5 impurities and limited according to ICH Q3A (R2) and B (R2) (2006) guidelines. However, certain regulatory authorities raised the concern that the Ames test may not be sufficiently sensitive to detect a mutagenic potential of nitrosamines and requested a confirmatory in vivo study using a transgenic animal genotoxicity model. Our data show that 181-14 was not mutagenic in the transgenic gene mutation assay in MutaTMMice. The data support the conclusion that the Ames test is an adequate and sensitive test system to assess a mutagenic potential of nitrosamines.

Stability-indicating reversed-phase-HPLC method development and validation for sacubitril/valsartan complex in the presence of impurities and degradation products: Robustness by quality-by-design approach.

According to current regulatory guidelines, a stability-indicating method has been developed to determine the impurities in sacubitril (SCB) and valsartan (VLS) tablet dosage forms and perform robustness studies using the design of experiments approach. The present study was initiated to understand quality target product profile, analytical target profile, and risk assessment for method variables that affect the method response. A reversed-phase-HPLC system was equipped with a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 µm) and a photo diode array detector. A gradient mobile phase was used in this research work. The detection was performed at 254 nm; the flow rate was 1.5 mL/min, and the column temperature was maintained at 30°C. The proposed method was validated per the International Council for Harmonisation Q2 (R1) guidelines. The coefficient of correlation was >0.999 for all impurities. The limits of detection and quantification were evaluated for SCB, VLS, and all impurities. The precision and accuracy were obtained for SCB, VLS, and their related impurities. Intra- and inter-day relative standard deviation values were less than 10.0%, and the recoveries of impurities varied between 90.0 and 115.0%. Based on the validation results, the proposed DoE method can estimate SCB and VLS impurities in the finished dosage form.

Application of capillary electrophoresis-nano-electrospray ionization-mass spectrometry for the determination of N-nitrosodimethylamine in pharmaceuticals.

After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.

Preparation of magnetic molecularly imprinted polymer for dispersive solid-phase extraction of valsartan and its determination by high-performance liquid chromatography: Box-Behnken design.

In this work, core/shell magnetic molecularly imprinted polymer nanoparticles were synthesized for extraction and pre-concentration of valsartan from different samples and then it was measured with high-performance liquid chromatography. For preparation of molecularly imprinted polymer nanoparticles, Fe3 O4 nanoparticles were coated with tetraethyl orthosilicate and then functionalized with 3-(trimethoxysilyl) propyl methacrylate. In the next step, molecularly imprinted polymer nanoparticles were synthesized under reflux and distillation conditions via polymerization of methacrylic acid, valsartan (as a template), azobisisobutyronitrile and ethylene glycol dimethacrylate as cross linking. The properties of molecularly imprinted polymer nanoparticle were investigated by FTIR spectroscopy, field emission scanning electron microscopy, and X-ray diffraction. Box-Behnken design with the aid of desirability function was used for optimizing the effect of variables such as the amounts of molecularly imprinted polymer nanoparticles, time of sonication, pH, and volume of methanol on the extraction percentage of valsartan. According to the obtained results, the affecting variables extraction condition were set as 10 mg of adsorbent, 16 min for sonication, pH = 5.5 and 0.6 mL methanol. The obtained linear response (r2  > 0.995) was in the range of 0.005-10 µg/mL with detection limit 0.0012 µg/mLand extraction recovery was in the range of 92-95% with standard deviation less than 6% (n = 3).

Rapid and efficient high-performance liquid chromatography analysis of N-nitrosodimethylamine impurity in valsartan drug substance and its products.

In July 2018, certain valsartan-containing drugs were voluntary recalled in Japan owing to contamination with N-nitrosodimethylamine (NDMA), a probable human carcinogen. In this study, an HPLC method was developed for the quantitative detection of NDMA simultaneously eluted with valsartan. Good linearity with a correlation coefficient (R2) > 0.999 was achieved over the concentration range of 0.011-7.4 µg/mL. The limits of detection and quantification were 0.0085 µg/mL and 0.0285 µg/mL, respectively. When the recalled valsartan samples were subjected to this method, the observed NDMA contents were in agreement with the reported values, indicating that our method achieved sufficient linearity, accuracy, and precision to detect NDMA in valsartan drug substances and products. Moreover, six samples (valsartan drug substances and tablet formulations), which had a possibility for NDMA contamination, were analyzed; none of the samples contained NDMA at detectable levels. Our method would be useful for the rapid screening and quantification of NDMA impurity in valsartan drug substances and products.

A new strategy for taste masking on bitter drug by other combined drug in fixed-dose combination: bitterness of Amlodipine besylate could be masked efficiently by Valsartan.

OBJECTIVES: The aim of this study was to evaluate the bitterness of amlodipine besylate (AML) combined with other five antihypertensive drugs: alacepril, benazepril, hydrochlorothiazide, telmisartan (TEL) and valsartan (VAL), which have possibility of usage as a fixed-dose combination (FDC) drugs. METHODS: The bitterness of individual six drugs and AML combined with each of the five drugs was evaluated using taste sensor SA402B (Intelligent Sensor Technology Inc.). AML combined with TEL or VAL was evaluated by taste sensor and human gustatory sensation tests. The interaction between AML with TEL or VAL was evaluated by 1 H-NMR. KEY FINDINGS: The bitterness of AML was significantly decreased by addition of VAL, whereas it remained unchanged by the addition of TEL in taste sensor and human gustatory sensation test. In the 1 H-NMR spectrum of AML with VAL, signal shifts of protons in AML were observed compared to that in AML alone. On the other hand, in the 1 H-NMR spectrum of AML with TEL, signal shifts of protons in AML were not observed. CONCLUSIONS: It was suggested that when VAL was mixed with AML, the electrostatic interactions between positive charged amino group of AML and negative charged tetrazole group of VAL were caused, and thereby led the suppression the bitterness of AML.

The contamination of valsartan and other sartans, part 1: New findings.

In July 2018 one of the bestselling antihypertensive agents valsartan manufactured in China was found to be contaminated by the "probably carcinogenic" nitrosamine N-nitrosodimethylamine (NDMA), followed by the detection of N-nitrosodiethylamine (NDEA) by us and others soon after. Our work also revealed that two additional non-nitrosamine contaminations valeramide (VLA) and N,N-dimethylvaleramide (VLA-DEM) were present in sartan tablets. Early measurements by others and us were performed by GC-MS or GC-MS/MS, which does not reach the sensitivity needed to find and quantitate trace levels of NDMA and NDEA. A highly sensitive LC-MS/MS method with APCI ionization was developed to detect and quantitate NDMA, NDEA, VLA and VLA-DIM in 152 sartan tablets from 8 structurally different sartan molecules. Good linearity for each compound could be demonstrated over calibration ranges in the lower nanograms. The assay for all substances was accurate and precise. With this method, a LLOQ of 0.00026 ppm for NDMA and 0.00013 ppm for NDEA could be achieved. NDMA, NDEA, VLA and VLA-DIM were found in 21 (13.8%), 9 (5.9%), 13 (8.6%) and 7 (4.6) % of the tablets, respectively. In addition, one candesartan product was found contaminated with NDEA. The implications of our findings for the testing of pharmaceutical products are discussed.

Lesson Learnt from Recall of Valsartan and Other Angiotensin II Receptor Blocker Drugs Containing NDMA and NDEA Impurities.

The presence of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities in angiotensin II receptor blocker (ARB) drugs containing tetrazole ring has triggered worldwide product recalls. The purpose of this article is to identify the potential gap area in current pharmaceutical industry practice that might have led to the NMDA and NDEA impurities escaping the drug manufacturer's and FDA's attention. The impact of process change was not adequately assessed by the manufacturer of contaminated APIs (active pharmaceutical ingredients), and potential for generation of mutagenic or other toxic impurities was not considered. The safety and risk associated with a chemical synthetic process was also not evaluated. This is primarily due to current industry practice which focuses on controlling the impurities above reporting threshold. ICH Q3A and FDA guidance on genotoxic and carcinogenic impurities in drug substances and products need to be integrated so that the ICH Q3A decision tree (attachment 3) begins by checking whether the synthetic process has been evaluated for the potential to generate toxic impurities. The compliance with ICH Q3A limits should be carried out only after the process has been determined to be safe without the risk of generating mutagenic and carcinogenic impurities.

Short commentary on NDMA (N-nitrosodimethylamine) contamination of valsartan products.

A range of generic valsartan products have been found to be contaminated with nitrosamines (principally N-nitrosodimethylamine; NDMA). We present information and discuss various elements of this phenomenon including: actions taken by regulatory agencies, source of the nitrosamine impurities, range of possible risk assessments based mainly on ICH M7 criteria, epidemiological assessment and analytical aspects.

Advanced oxidation of antihypertensives losartan and valsartan by photo-electro-Fenton at near-neutral pH using natural organic acids and a dimensional stable anode-gas diffusion electrode (DSA-GDE) system under light emission diode (LED) lighting.

In this work photo-electro-Fenton (PEF) processes using a dimensionally stable anode-gas diffusion electrode (DSA-GDE) system under light emission diodes (LED)-type radiation were used in the degradation of the angiotensin-II-receptor antagonists (ARA II), valsartan (VAL), and losartan (LOS), which are used in the treatment of hypertension diseases, and are considered among the emerging contaminants (ECs). Organic acids as citric, tartaric, and oxalic acids were used as complexing agents of iron ions in order to maintain the performance of the Fenton reaction at near-neutral pH value. The results show that at 3.42 mA/cm2 after 90 min of electro-Fenton (EF) treatment, degradation of 70% of VAL and 100% of LOS were observed. Total degradation of VAL and LOS was reached with a PEF process at the same time with mineralization of 30%. When citric and tartaric acids were used instead of oxalic acid, similar results were obtained, i.e., total degradation of both compounds, LOS and VAL, after 90 min of treatment. The degradation performance can be attributed to the increase of the initial dissolved iron in the system, facilitating the Fe3+/Fe2+ turnover in the catalytic photo-Fenton reaction and consequently, hydroxyl radical (•OH) production. In addition, the increased photo-activity of the complexes can be associated with their high capability to complex Fe3+ and to promote ligand-to-metal charge transfer, which is of key importance to feed Fe2+ to the Fenton process. The results show that the system evaluated was more efficient to eliminate sartan family compounds using LED lighting in comparison with traditional UV-A lamps used in this kind of work. Moreover, three transformation products of VAL degradation and two transformation products of LOS degradation were identified by high-resolution mass spectrometry (HRMS) using hybrid quadrupole-time-of-flight (QTOF) MS and, at the end of the PEF system, the several organic compounds accumulated and no mineralized were effectively treated in a subsequent aerobic biological system.

Transfer Behavior of the Weakly Acidic BCS Class II Drug Valsartan from the Stomach to the Small Intestine During Fasted and Fed States.

The objective of this study was to investigate the transfer behavior of the weakly acidic BCS class II drug valsartan from the stomach to the small intestine during fasted and fed states. An in vitro transfer model previously introduced by Kostewicz et al. (J Pharm Pharmacol 56(1):43-51, 2004) based on a syringe pump and a USP paddle apparatus was used to determine the concentration profiles of valsartan in the small intestine. Donor phases of simulated gastric fluid during fasted (FaSSGF) and fed (FeSSGF) states were used to predisperse Diovan® tablets (160 mg valsartan). The initial concentrations of valsartan in FaSSGF and FeSSGF were 6.2 and 91.8%, respectively. Valsartan dispersions were then transferred to acceptor phases that simulate intestinal fluid and cover the physiological properties (pH, buffer capacity, and ionic strength) of the gastrointestinal fluid at a flow rate of 2 mL/min. The pH measurements were reported at time intervals corresponded to those of the transfer experiments to investigate the effect of percent dissolved of valsartan in the donor phase on lowering the pH of the acceptor phases. The f2 similarity test was used to compare the concentration profiles in the acceptor phases. In fasted state, the concentration of valsartan in the acceptor phases ranged between 33.1 and 89.4% after 240 min. Whereas in fed state, valsartan was fully dissolved in all acceptor phases within a range of 94.5-104.9% after 240 min. Therefore, the transfer model provides a useful screen for the concentrations of valsartan in the small intestine during fasted and fed states.

Chronotherapeutically Modulated Pulsatile System of Valsartan Nanocrystals-an In Vitro and In Vivo Evaluation.

The objective was to improve the dissolution of valsartan by developing valsartan nanocrystals and design a pulsed release system for the chronotherapy of hypertension. Valsartan nanocrystals were prepared by sonication-anti-solvent precipitation method and lyophilized to obtain dry powder. Nanocrystals were directly compressed to minitablets and coated to achieve pulsatile valsartan release. Pharmacokinetic profiles of optimized and commercial formulations were compared in rabbit model. The mean particle size and PDI of the optimized nanocrystal batch V4 was reported as 211 nm and 0.117, respectively. DSC and PXRD analysis confirmed the crystalline nature of valsartan in nanocrystals. The dissolution extent of valsartan was markedly enhanced with both nanocrystals and minitablets as compared to pure valsartan irrespective of pH of the medium. Core minitablet V4F containing 5% w/w polyplasdone XL showed quickest release of valsartan, over 90% within 15 min. Coated formulation CV4F showed two spikes in release profile after successive lag times of 235 and 390 min. The pharmacokinetic study revealed that the bioavailability of optimized formulation (72.90%) was significantly higher than the commercial Diovan tablet (30.18%). The accelerated stability studies showed no significant changes in physicochemical properties, release behavior, and bioavialability of CV4F formulation. The formulation was successfully designed to achieve enhanced bioavailability and dual pulsatile release. Bedtime dosing will more efficiently control the circadian spikes of hypertension in the morning.

New methods for amlodipine and valsartan native spectrofluorimetric determination, with factors optimization study.

Four native fluorescence methods were suggested for simultaneous determination of amlodipine (AML) and valsartan (VAL). These methods were based on excitation of both drugs at λ(ex) 300 nm, in one step, to give maximum emission at λ(em) 378 and 496 nm for AML and VAL, respectively. The first method, single λ(ex) method, was used without any additions. The sensitivity of this method was further increased by the addition of hydroxy propylmethyl cellulose (HPMC) surfactant, ß-cyclodextrin, or ferric oxide magnetite nanoparticles, in the other three methods. Different types of surfactants, and different concentration levels of both ß-cyclodextrin and ferric oxide nanoparticles, were scanned to determine the optimum conditions for enhancing the sensitivity. Some factors affecting the fluorescence intensity of both cited drugs, like the type and volume of the added solvent (to be used as a sensing agent), and pH of measurement were studied and optimized. The proposed methods could be used in determination of AML and VAL in bulk powder, their laboratory prepared mixtures and pharmaceutical formulations. The obtained results were statistically compared to each other and to that of some reported methods. The specificity of the developed methods was investigated, and the methods were validated according to ICH guidelines.