Commercial Pharmaceutical Products Research Articles

Measurement uncertainty of lactase-containing tablets analyzed with FTIR

Uncertainty is one of the most critical aspects in determination of measurement reliability. In order to ensure accurate measurements, results need to be traceable and uncertainty measurable. In this study, homogeneity of FTIR samples is determined with a combination of variographic and multivariate approach. An approach for estimation of uncertainty within individual sample, as well as, within repeated samples is introduced. FTIR samples containing two commercial pharmaceutical lactase products (LactaNON and Lactrase) are applied as an example of the procedure.The results showed that the approach is suitable for the purpose. The sample pellets were quite homogeneous, since the total uncertainty of each pellet varied between 1.5% and 2.5%. The heterogeneity within a tablet strip was found to be dominant, as 15–20 tablets has to be analyzed in order to achieve <5.0% expanded uncertainty level. Uncertainty arising from the FTIR instrument was <1.0%. The uncertainty estimates are computed directly from FTIR spectra without any concentration information of the analyte.

Electropolymerized Supramolecular Tetraruthenated Porphyrins Applied as a Voltammetric Sensor

Porphyrin 5,10,15,20-Tetra(4-pyridyl)manganese(III), [Mn-TPyP(H2O)2]PF6, and electropolymerized supramolecular porphyrins (ESP), {Mn-TPyP(H2O)2[RuCl3(dppb)]4}PF6 (dppb = 1,4-bis(diphenilphosphine)butane), were synthesized and characterized. A thin solid film of ESP was obtained on a glass carbon electrode surface by a cyclic voltammetry method. The peak current increased with the number of voltammetric cycles, which shows a typical behavior of the species being adsorbed on the surface of the electrode. Cyclic voltammetry was also employed for acetaminophen quantification using an ESP modified electrode. The modified electrode shows a linear relationship between the anodic peak current and the concentration of acetaminophen (in the rage 0.05 to 0.7 mmol L - 1). The performance of the modified electrode was verified by the determination of acetaminophen in a commercial pharmaceutical product and the results were in good agreement with those obtained by a control HPLC method.

Translating Nanotechnology from Bench to Pharmaceutical Market: Barriers, Success, and Promises

Nanotechnology is a buzzword of this millennium and it has transformed the face of research in science and technology. The advent of nanotechnology has also influenced the biomedical and pharmaceutical research since last decade. Various nano-architectures have been designed for improving the therapeutic performance of drugs, proteins, peptides, and genes and to achieve their targeting at the site of action. Although nanotechnology has demonstrated dramatic potential in drug delivery research, like any technology, its real success depends on the ability of drug delivery scientists to translate and scale innovations to the commercial pharmaceutical products. It is indeed a very challenging task to successfully overcome manufacturing, clinical, and regulatory hurdles associated with a nanotech product. Nevertheless, the pharmaceutical industry has witnessed commercialization of the nanotechnology-based products for various applications. In the present special issue, we have tried to consolidate various aspects of existing and upcoming nanotechnologies for drug delivery. Contribution by V. Morigi et al. takes an overview of business potential and market trend of pharmaceutical nanotechnology. The authors have also discussed financial aspects of nanotechnology by citing noteworthy examples of few nanotech products that have already been commercialized. This contribution could be useful to scientists aiming to start up nanotechnological business ventures. Contribution by N. Anton et al. demonstrates how nanotechnology can change the face of conventional drug delivery systems. In this interesting investigation, the authors demonstrate that coating of conventional tablets with lipid nanoemulsion can be used to modulate the release of the drug from tablet matrix. The paper by P. Severino et al. gives an account of potential of solid lipid nanocarriers for the oral delivery of drugs and peptides. The authors have provided information about the lipids that can be used for oral delivery, role of lipids in the oral delivery, toxicological aspects of lipid nanocarriers, and products under clinical development. S. Banerjee et al. have given a complete overview of polyethylene-glycol- (PEG-) based conjugates for drug delivery. The contribution fosters understanding design aspects of and chemistry behind PEG-based nano-architectures for drug delivery. Furthermore, the paper has a detailed discussion on the various PEG-conjugates available in the pharmaceutical market. Contribution by A. Garcia et al. highlights the potential of particle replication in nonwetting templates (PRINT), a platform technology based on lithographic techniques for drug delivery applications. The contribution clearly demonstrates potential of PRINT technology to generate particles of various, but precise, morphology for a variety of drugs and biotechnology-based therapeutics (proteins and siRNA). The application of PRINT technology for generating aerosols for pulmonary applications has also been described. This contribution is an example of the attributes required from a nanofabrication technique to circumvent manufacturing-related issues in pharmaceutical nanotechnology. The paper by F. Lallemand et al. delineates various aspects of and challenges in ocular drug delivery and systematically describes development of Novasorb, a cationic nanoemulsion-based platform ocular delivery system. The authors have furnished a detailed description of the formulation development aspects and ocular safety of excipients which is followed by in vivo proof-of-concept and clinical development. This paper gives clear insight into various challenges faced for developing nanomedicine for ocular delivery. The paper by J. D. Heidel and T. Schleup throws light on the various applications of self-assembled nanocarriers consisting of cyclodextrin based polymers. The authors describe various developmental aspects of two platforms based on cyclodextrin-based polymers (Cyclosert and RONDEL) which can enable efficient delivery of drugs or nucleic-acid-based therapeutics. The translational aspects of both the nanocarriers and in vivo proof-of-concept have also been furnished in this paper. The research paper by J. Rios-Doria et al. gives insight into developmental aspects of a pH sensitive cross-linked polymeric micelle technology (IVECT). The authors describe synthesis of the polymeric micelles, their ability to encapsulate various drugs, and in vivo proof-of-concept for anticancer drugs like daunorubicin and BB4007431. In a nutshell, we believe that this special issue would give readers insight into various aspects involved in translating nanotechnology from bench to pharmaceutical market. Moreover, the special issue also includes some contributions about nanotechnologies that are currently under clinical development. Abhijit A. Date Rajesh R. Patil Riccardo Panicucci Eliana B. Souto Robert W. Lee

Quality Considerations in the Establishment of Specifications for Pharmaceuticals

The quality of a commercial pharmaceutical product is set by a well-designed, understood, and executed manufacturing process using high-quality raw materials. In addition, this built-in quality is verified at release and its stability is assured by random sampling and testing of critical product characteristics against established specifications. There are several approaches to how this verification testing is designed, depending on the underlying quality philosophy employed. This article explores different common approaches and briefly discusses the pros and cons of each. In the most general sense, quality can be defined as the extent to which a product meets customer expectation. The common practice in the pharmaceutical industry is to specify test acceptance criteria without clearly defining or relating these criteria to an underlying quality statement about the product. Fundamental to constructing such a quality statement is an assumed definition of what is meant by drug product in this context, that is, batch, individual dosage units, or some other basic unit. As a result, it is not always fully clear what a reasonable customer expectation is. In contrast to current practices, we propose that a transparent quality statement should be formulated for every pharmaceutical product and that the test(s) to verify that the required performance (as defined by the quality statement) should be designed using established statistical techniques.

Simultaneous Determination of Ritonavir and Atazanavir in Combined Tablet Dosage Form by HPTLC

A new, simple, sensitive, precise and accurate High-performance thin-layer chromatographic method for simultaneous determination of Ritonavir and Atazanavir in their combined tablet dosage form has been developed, validated and used for determination of the compounds in commercial pharmaceutical products. Chromatographic separation was achieved on aluminium plates precoated with silica gel 60 F254 as the stationary phase and chloroform: ethyl acetate: acetone (5:2:3, v/v/v) as a mobile phase. Densitometric measurements of their spots were achieved at 244 nm over the concentration ranges of 800-2800 ng spot-1 and 2400-8400 ng spot-1, with mean recoveries of 98.57 ± 0.35 and 99.16 ± 0.20 for ritonavir and atazanavir respectively. Limit of detection for ritonavir and atazanavir were found to be 300 ng spot-1 and 200 ng spot-1 respectively.

Antitumor activity of mushroom polysaccharides: a review

Mushrooms were considered as a special delicacy by early civilizations and valued as a credible source of nutrients including considerable amounts of dietary fiber, minerals, and vitamins (in particularly, vitamin D). Mushrooms are also recognized as functional foods for their bioactive compounds offer huge beneficial impacts on human health. One of those potent bioactives is β-glucan, comprising a backbone of glucose residues linked by β-(1→3)-glycosidic bonds with attached β-(1→6) branch points, which exhibits antitumor and immunostimulating properties. The commercial pharmaceutical products from this polysaccharide source, such as schizophyllan, lentinan, grifolan, PSP (polysaccharide-peptide complex) and PSK (polysaccharide-protein complex), have shown evident clinical results. The immunomodulating action of mushroom polysaccharides is to stimulate natural killer cells, T-cells, B-cells, neutrophils, and macrophage dependent immune system responses via differing receptors involving dectin-1, the toll-like receptor-2 (a class of proteins that play a role in the immune system), scavengers and lactosylceramides. β-Glucans with various structures present distinct affinities toward these receptors to trigger different host responses. Basically, their antitumor abilities are influenced by the molecular mass, branching configuration, conformation, and chemical modification of the polysaccharides. This review aims to integrate the information regarding nutritional, chemical and biological aspects of polysaccharides in mushrooms, which will possibly be employed to elucidate the correlation between their structural features and biological functions.

Quality assessment and origin tracing of Guangdong Liangcha granules using direct mass spectrometry fingerprinting

A novel fingerprinting approach for quality assessment and origin tracing of Guangdong Liangcha granules (GLG) by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) has been established. A negative-ion ESI-TOF spectrum gave the best chemical information and was most suitable to construct the chemical profile fingerprinting of GLG. Using high-resolution TOF-MS, 61 common mass spectral peaks based on the major constituents present in GLG were accurately and simultaneously detected without any chromatographic separation, providing valuable information for overall assessment of the quality and differentiation of the origin of the preparation. Six sources of commercial GLG products produced by five different manufacturers have been successfully differentiated, according to the principal component analysis of 61 common mass spectral peaks, and the stability of them was also assessed. The results of our experimental data demonstrated that ESI-TOF-MS fingerprinting is a useful tool for rapid pharmaceutical analysis, with promising prospects for overall quality assessment and origin differentiation of commercial pharmaceutical products.

Treatment of Acute Diarrhea With Saccharomyces boulardii in Infants

The aim of the study was to determine whether an oral treatment with a commercial pharmaceutical product containing Saccharomyces boulardii would reduce the duration of diarrhea in infants with acute diarrhea. In the present double-blind, placebo-controlled study, 186 infants, 6 to 48 months old and hospitalized within 72 hours after the onset of acute diarrhea in 2 hospitals in Goiânia, Goiás, Brazil, were randomly assigned to receive twice per day for 5 days 200 mg of a commercial pharmaceutical product containing 4 × 10 viable cells of S boulardii or a placebo. Stool samples were submitted to search for rotavirus. Among the 176 infants who completed the trial, those treated with S boulardii (90) showed a reduction in diarrhea duration (P < 0.05) when compared with the placebo group (86). The present study shows a reduction in diarrhea duration when S boulardii was given to children within 72 hours after the onset of acute diarrhea. The present study suggests a complementary treatment of acute diarrhea in infants with daily oral doses of S boulardii.

Attenuated total reflectance with Fourier transform infrared spectroscopy (ATR/FTIR) and different PLS Algorithms for simultaneous determination of clavulanic acid and amoxicillin in powder pharmaceutical formulation

A method for simultaneous determination of clavulanic acid (CA) and amoxicillin (AMO) was developed using Fourier transform mid infrared technique coupled with attenuated total reflectance (ATR/FTIR) accessory. 27 samples were used as calibration set and 8 samples were used for prediction set. Calibration models were developed using partial least squares (PLS), interval PLS (iPLS), synergy PLS (siPLS) and backward PLS (biPLS). Multiplicative scatter correction and the mean centering were used and produced the best models. Relative standard error of prediction (RSEP) of 3.8% for CA and 5.1% for AMO were obtained using biPLS algorithm for ATR/FTIR data. Results obtained by the proposed methodology were compared with those using high performance liquid chromatography (HPLC) and no significant differences were obtained. The proposed method using ATR/FTIR combined to multivariate analysis methods was suitable for the simultaneous determination of CA and AMO in commercial pharmaceutical products.

The influence of dissolution conditions on the drug ADME phenomena

In this work, a review of the apparatuses available to mimic what happens to a drug (or to foodstuffs) once ingested is presented. Similarly, a brief review of the models proposed to simulate the fate of a drug administered to a living body is reported. Then, the release kinetics of extended release of diclofenac from a commercial enteric-coated tablet was determined both in a conventional dissolution tester (USP Apparatus 2, Method A) as well as in an apparatus modified to reproduce a given pH evolution, closer to the real one than the one suggested by USP. The two experimental release profiles were reported and discussed; therefore, they were adopted as input functions for a previously proposed pharmacokinetic model. The obtained evolutions with time of plasma concentration were presented and used to assess the effectiveness of the commercial pharmaceutical products. The importance of a correct in vitro simulation for the design of pharmaceutical dosage systems was thus emphasized.

Determination of ciclopirox olamine in pharmaceutical products by capillary electrophoresis with capacitively coupled contactless conductivity detection

This paper describes the determination of ciclopirox olamine in pharmaceutical formulations using capillary electrophoresis with capacitively coupled contactless conductivity detection. In an alkaline medium, ciclopirox olamine is converted into an anionic species and its detection is possible in capillary electrophoresis with capacitively coupled contactless conductivity detection without an electroosmotic flow modifier, because it is a low-mobility species. A linear working range from 2.64 to 264 μg/mL in sodium hydroxide electrolyte as well as low detection limit (0.39 μg/mL) and a good repeatability (RSD = 3.4% for 264 μg/mL ciclopirox solution (n = 10)) were achieved. It was also possible to determine olamine in its cationic form when acetic acid was used as the electrolyte solution. The results obtained include a linear range from 26.4 to 184.8 μg/mL and a detection limit of 2.6 μg/mL olamine. The proposed methods were applied to the analysis of commercial pharmaceutical products and the results were compared with the values indicated by the manufacturer as well as those obtained using a titrimetric method recommended by American Pharmacopoeia.

Quantification of N-acetylcysteine in pharmaceuticals using cobalt phthalocyanine modified graphite electrodes

Flow injection analysis (FIA) with amperometric detection was employed for the quantification of N-acetylcysteine (NAC) in pharmaceutical formulations, utilizing an ordinary pyrolytic graphite (OPG) electrode modified with cobalt phthalocyanine (CoPc). Cyclic voltammetry was used in preliminary studies to establish the best conditions for NAC analysis. In FIA-amperometric experiments the OPG–CoPc electrode exhibited sharp and reproducible current peaks over a wide linear working range (5.0×10−5–1.0×10−3molL−1) in 0.1molL−1 NaOH solution. High sensitivity (130mAmol−1cm2) and a low detection limit (9.0×10−7molL−1) were achieved using the sensor. The repeatability (R.S.D.%) for 13 successive flow injections of a solution containing 5.0×10−4molL−1 NAC was 1.1%. The new procedure was applied in analyses of commercial pharmaceutical products and the results were in excellent agreement with those obtained using the official titrimetric method. The proposed amperometric method is highly suitable for quality control analyses of NAC in pharmaceuticals since it is rapid, precise and requires much less work than the recommended titrimetric method.

Tracing Origins of Complex Pharmaceutical Preparations Using Surface Desorption Atmospheric Pressure Chemical Ionization Mass Spectrometry

A novel strategy to trace the origins of commercial pharmaceutical products has been developed based on the direct chemical profiling of the pharmaceutical products by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS). Besides the unambiguous identification of active drug components, various compounds present in the matrixes are simultaneously detected without sample pretreatment, providing valuable information for drug quality control and origin differentiation. Four sources of commercial amoxicillin products made by different manufacturers have been successfully differentiated. This strategy has been extended to secerning six sources of Liuwei Dihuang Teapills, which are herbal medicine preparations with extremely complex matrixes. The photolysis status of chemical drug products and the inferior natural herd medicine products prepared with different processes (e.g., extra heating) were also screened using the method reported here. The limit of detection achieved in the MS/MS experiments was estimated to be 1 ng/g for amoxicillin inside the capsule product. Our experimental data demonstrate that DAPCI-MS is a useful tool for rapid pharmaceutical analysis, showing promising perspectives for tracking the entire pharmaceutical supply chain to prevent counterfeit intrusions.

Ion Beam Analysis and PD-MS As New Analytical Tools for Quality Control of Pharmaceuticals: Comparative Study from Fluphenazine in Solid Dosage Forms

In order to evaluate the potential of accelerator based analytical techniques ((particle induced X-ray emission (PIXE), particle induced gamma-ray emission (PIGE), and particle desorption mass spectrometry (PD-MS)) for the analysis of commercial pharmaceutical products in their solid dosage form, the fluphenazine drug has been taken as a representative example. It is demonstrated that PIXE and PIGE are by far the best choice for quantification of the active ingredient (AI) (certification with 7% precision) from the reactions induced on its specific heteroatoms fluorine and sulfur using pellets made from original tablets. Since heteroatoms cannot be present in all types of drugs, the PD-MS technique, which makes easily the distinction between AI(s) and excipients, has been evaluated for the same material. It is shown that the quantification of AI is obtained via the detection of its protonated molecule. However, calibration curves have to be made from the secondary ion yield variations since matrix effects of various nature are characteristics of such mixtures of heterogeneous materials (including deposits from soluble components). From the analysis of solid tablets, (either transformed into pellets and even as received), it is strongly suggested that the physical state of the grains in the mixture is a crucial parameter in the ion emission and accordingly for the calibration curves. As a result of our specific (but not optimized) conditions the resulting precision is <17% with an almost linear range extending from 0.04 to 7.87 mg of AI in a tablet made under the manufacturer conditions (the commercial drug product is labeled at 5 mg).

Simultaneous Spectrophotometric Determination of Aspirin, Paracetamol, Caffeine, and Chlorphenamine from Pharmaceutical Formulations Using Multivariate Regression Methods

This paper presents a simultaneous spectrophotometric determination of aspirin, paracetamol, caffeine, and chlorphenamine from commercial pharmaceutical products using principal component regression and partial-least squares regression. The concentration of the training set was established employing a partial factorial calibration design at four levels. Several quality parameters and recovery values obtained on authentic samples illustrated excellent performance characteristics concerning the goodness of fit and the accuracy and precision of prediction. Eight pharmaceutical formulations containing at least two of these four mentioned active ingredients and diverse electuaries were successfully analyzed. The obtained results were also validated by high-performance liquid chromatography.

Electrooxidation of dextromethorphan on a carbon nanotube–carbon microparticle–ionic liquid composite: applied to determination in pharmaceutical forms

The electrooxidation of dextromethorphan on a composite constructed with carbon nanotube–ionic liquid–carbon microparticles was investigated by cyclic voltammetry in a 100 mM phosphate buffer solution, pH 7.40. In the voltammograms, an irreversible diffusion-controlled anodic peak appeared. The diffusion coefficient of dextromethorphan, the electron-transfer coefficient, and the standard rate constant of the electrooxidation process were found to be 3.45 × 10−6 cm2 s−1, 0.65, and 1.67 × 10−3 cm s−1, respectively. A sensitive and timesaving determination procedure was developed for the analysis of dextromethorphan, and the corresponding analytical parameters were reported. Using this method, dextromethorphan was determined with an LOD and LOQ of 8.81 and 29.36 μM in a linear range of 2.5 × 10−4 to 3.3 × 10−3 M, respectively. The proposed amperometric method was successfully applied to the analysis of commercial pharmaceutical products (syrup and oral drop), and the results were in good agreement with the declared values.

Electrocatalytic oxidation of the antiviral drug acyclovir on a copper nanoparticles-modified carbon paste electrode

The electrocatalytic oxidation of acyclovir (Zovirax) on two different copper-based electrodes: copper microparticles- and copper nanoparticles-modified carbon paste electrodes (denoted as micro-CPE and nano-CPE, respectively) was voltammetrically investigated. In the voltammogram recorded using micro-CPE, a single anodic oxidation peak appeared, while nano-CPE resulted in two overlapped anodic peaks. The anodic currents were related to the electrocatalytic oxidation of acyclovir via the electrogenerated active species of Cu(III) with an EC’ mechanism. Acyclovir was oxidized with higher rates at low potentials on nano-CPE compared to micro-CPE. This was related to the nanosize effect of copper nanoparticles. The constants of the electrocatalytic oxidation process and the diffusion coefficient of acyclovir were reported. A sensitive and time-saving determination procedure was developed for the analysis of acyclovir and the corresponding analytical parameters were reported. The proposed amperometric method was applied to the analysis of commercial pharmaceutical products (tablets and topical cream) and the results were in good agreement with the declared values.

Determination of Epinephrine by Flow Injection Analysis Coupled Ag(III) Complex‐Luminol Chemiluminescence Detection

AbstractA new Ag(III) complex‐luminol chemiluminescent system which was applied to the determination of epinephrine is firstly reported. Based on the enhancing effect of epinephrine on the chemiluminescence reaction of luminol with [Ag(HIO6)2]5− in alkaline solution, a highly sensitive chemiluminescence (CL) detection by flow injection analysis (FIA) was developed for epinephrine. Under the optimum conditions, CL intensity was proportional to concentration of epinephrine in the 1.0×10−9–1.0×10−7 mol·L−1 range. The limit of detection was 8.0×10−10 mol·L−1 for epinephrine (3δ), with a relative standard deviation (n=11) of 2.9% for 1.5×10−8 mol·L−1 epinephrine. The method validation was done with epinephrine determinations in commercial pharmaceutical products. The mechanism of the reactions was also discussed.

Electrochemical study and flow injection analysis of paracetamol in pharmaceutical formulations based on screen-printed electrodes and carbon nanotubes

Acetaminophenol or paracetamol is one of the most commonly used analgesics in pharmaceutical formulations. Acetaminophen is electroactive and voltammetric mechanistic studies for the electrode processes of the acetaminophenol/N-acetyl-p-quinoneimine redox system are presented. Carbon nanotubes modified screen-printed electrodes with enhanced electron transfer properties are used for the study of the electrochemical–chemical oxidation mechanism of paracetamol at pH 2.0. Quantitative analysis of paracetamol by using its oxidation process (in a Britton–Robinson buffer solution pH 10.0) at +0.20 V (vs. an Ag pseudoreference electrode) on an untreated screen-printed carbon electrode (SPCE) was carried out. Thus, a cyclic voltammetric based reproducible determination of acetaminophen (R.S.D., 2.2%) in the range 2.5 × 10 −6 M to 1 × 10 −3 M, was obtained. However, when SPCEs are used as amperometric detectors coupled to a flow injection analysis (FIA) system, the detection limit achieved for paracetamol was 1 × 10 −7 M, one order of magnitude lower than that obtained by voltammetric analysis. The repeatability of the amperometric detection with the same SPCE is 2% for 15 successive injections of 10 −5 M acetaminophen and do not present any memory effect. Finally, the applicability of using screen-printed carbon electrodes for the electrochemical detection of paracetamol (i.e. for quality control analysis) was demonstrated by using two commercial pharmaceutical products.

Simultaneous determination of sulphamethoxazole and trimethoprim in powder mixtures by attenuated total reflection-Fourier transform infrared and multivariate calibration

A partial least-squares calibration (PLS) procedure in combination with infrared spectroscopy has been developed for simultaneous determination of sulphamethoxazole (SMZ) and trimethoprim (TMP) in raw material powder mixtures used for manufacturing commercial pharmaceutical products. Multivariate calibration modeling procedures, interval partial least squares (iPLS) and synergy partial least squares (siPLS), were applied to select a spectral range that provided the lowest prediction error in comparison to the full-spectrum model. The experimental matrix was constructed using 49 synthetic samples and 15 commercial samples. The considered concentration ranges were 400–900mgg−1 SMZ and 80–240mgg−1 TMP. Spectral data were recorded between 650 and 4000cm−1 with a 4cm−1 resolution by Fourier transform infrared spectroscopy coupled with attenuated total reflectance (ATR-FTIR) accessory. The proposed procedure was compared with conventional procedure by high performance liquid chromatography (HPLC) using 15 commercial samples containing SMZ and TMP. The results showed that PLS regression model combined to ATR-FTIR is a relatively simple, rapid and accurate procedure that could be applied to the simultaneous determination of SMZ and TMP in routine quality control of powder mixtures. A root mean square error of prediction (RMSEP) of 13.18mgg−1 for SMZ and 6.03mgg−1 for TMP was obtained after selection of better intervals by siPLS. Using the proposed procedure it is possible to analyze each sample in less than 3min considering two replicates (excluding the grinding step). Accuracy was checked by comparison to HPLC method and agreement better than 98.8% was achieved.