Development and validation of a liquid chromatographic method for determination of lacidipine residues on surfaces in the manufacture of pharmaceuticals

A high-performance liquid chromatographic (HPLC) method for the assay of lacidipine residues in swabs collected from various surfaces involved in drug manufacture is described. The swabbing procedure using two cotton swabs was validated applying a wipe test. An RP-HPLC method, developed to determine low quantities of the drug in the presence of its main impurities, was also validated. To remove drug residues from stainless steel and glass surfaces, the first cotton swab must be soaked preferably in acetonitrile whereas, on vinyl surfaces better results are obtained using methanol. The HPLC method selected involves a C12 column, at 40 degrees C, a mixture of acetonitrile-0.05 M ammonium acetate (88:12, v/v) as a mobile phase and UV detection at 282 nm. Recoveries obtained are strongly dependent on the type of surface tested, being higher on stainless steel. The surface material has also different influence on the drug stability. The method was validated over a range of 0.5-100 microg/400 cm2 and had a detection limit of 0.1 microg/400 cm2.     

Validation of the removal of acetylsalicylic acid. Recovery and determination of residues on various surfaces by high performance liquid chromatographic

The validation of a procedure to clean glass, vinyl and stainless steel surfaces that have been exposed to acetylsalicylic acid during its manufacture is described. The cleaning procedure using two cotton swabs moistened with the mobile phase was validated using a wipe-test and a high-performance liquid chromatography (HPLC) method developed to determine low quantities of the acid. The HPLC method involves an octadecylsilane column at 55°C, a mixture of water–acetonitrile–orthophosphoric acid (779:220:1, v/v) as mobile phase and detection at 226 nm. Recoveries of 86%, 90% and 94% were obtained from vinyl, glass and stainless steel plates respectively. The validation gave acceptable levels of sensitivity, recovery, precision and linearity.     

Determination of the van der Waals, electron donor and electron acceptor surface tension components of static Gram-positive microbial biofilms

A large number of studies have shown the influence of the physico-chemical properties of a surface on microbial adhesion phenomenon. In this study, we considered that the presence of a bacterial biofilm may be regarded as a “conditioning film” that may modify the physico-chemical characteristics of the support, and thus the adhesion capability of planktonic micro-organisms coming into contact with this substratum. In this context, we adapted a protocol for biofilm formation that allows, under our experimental conditions, contact angle measurements, the reference method to determine the energetic surface properties of a substratum. This made it possible to determine the van der Waals, electron acceptor and electron donor properties of static biofilms grown at 25°C on stainless-steel slides with six Gram-positive bacteria isolated in dairy plants. A variance analysis indicated significant effects (P<0.05) of the bacterial strains and of the physiological state of the micro-organisms (planktonic or sessile) on the contact angles. To link the energetic properties of the six biofilms with direct adhesion experiments, we measured the affinity of fluorescent carboxylate-modified polystyrene beads for the different biofilm surfaces. The results correlated best with the electron-acceptor components of the biofilm surface energies, stressing the importance of Lewis acid–base interactions in adhesion mechanisms.     

Structure and electronic structure of quasicrystal and approximant surfaces: a photoemission study

The structure and electronic structure of different high-symmetry surfaces of either quasicrystalline or approximant Al–Pd–Mn were studied by means of photoemission-based techniques such as X-ray photoelectron diffraction (XPD) and ultraviolet photoelectron spectroscopy. We find that the twofold (2f), 3f and 5f surfaces of icosahedral Al–Pd–Mn exhibit all the symmetry elements of the icosahedral non-crystallographic group. These XPD experiments can be modeled by single-scattering cluster calculations.

The bulk-terminated icosahedral or approximant surfaces are recovered after ion sputtering followed by annealing at T≈500–600 °C. A wealth of ordered surface phases (with different compositions) are found after sputtering and depending on the annealing temperature as, for example, a crystalline bcc multitwinned phase (for T<400 °C) or a stable decagonal quasicrystalline surface (for T>650 °C).

The icosahedral surfaces are characterised by a lowering of the density of states close to the Fermi edge, compatible with the opening of a pseudogap, as expected for a quasicrystal. The crystalline overlayers are characterised by a sharp Fermi edge, while the approximant and decagonal quasicrystalline surfaces also have a lowered density of states.     

Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52

The polysaccharides associated with free (planktonic) and surface-attached (biofilm) cells from cultures of Pseudomonas fluorescens strain B52 were compared. Variations in the attached matrix due to surface material (glass or stainless steel) were also analyzed. Two digestion methods were used to optimize the recoveries of sugars, uronic acids and acidic substituents. The yield of analyzable material after digestion reached 90% for the material associated to the biofilms, though only 20–30% for that bound to planktonic cells. The polysaccharide(s) in the biofilm had glucuronic and guluronic acids as main components, besides rhamnose, glucose and glucosamine. The proportion of glucuronic to guluronic acid was higher in the polysaccharide(s) found in biofilms formed on stainless steel than in those on glass.     

A biofilm resistance surface yielded by grafting of antimicrobial peptides on stainless steel surface

Anti‐biofilm formation on the surface is a severe issue in medical implants, hull surface, and food industry. Antimicrobial peptide, magainin II, was covalently bound to stainless steel surfaces through multi‐step modification. The untreated and modified samples were analyzed by SEM‐EDS, XPS, and contact angle, respectively, which indicated the peptide was immobilized on the surfaces. The antimicrobial tests of modified samples were conducted using Staphylococcus aureus and Escherichia coli, and the results revealed that peptide modified surface decreased the biofilm and bacteria quantity of stainless steel surface.     

Effect of dose rate and comparison of different media used in the recuperation of gamma irradiated listeria monocytogenes

D₁₀ of different strains of Listeria monocytogenes (LM) gamma irradiated with varying dose-rates are were established. Our results seem to indicate that the dose-rate does not exert a significant effect on their D₁₀. D₁₀ obtained with LM irradiated and plated on Oxford agar showed the highest value.     

3-Bromo-2-methyl-1-propene: molecular structure and conformation in the gas phase as determined by electron diffraction and molecular mechanics calculation

A gas phase electron diffraction study of 3-bromo-2-methyl-1-propene shows that there is predominantly a gauche conformer present. Data recorded at 20 and 180°C show 4(8) and 5(4)% respectively of a second confomer with a planar heavy atom skeleton. The gauche structural results in terms of r_a distances and angles at 20°C were found to be: r(C---C) = 1.331(9) Å, r(C---CH₂Br) = 1.484(6) Å, r(C---CH₃) — r(C---CH₂Br) = 0.017 Å, (assumed), r(C---Br) = 1.965(6) Å, C=C---CH₂Br = 121.5(0.7)°, C=C---CH₂Br — C=C---CH₃ = 0.7° (constraint from molecular mechanics calculation), C---C---Br = 112.2(0.5)°, torsional ANGLE = 112.5(2.2)°. Uncertainties are given as 2σ, where σ includes uncertainties due to correlation among observations, electron wavelength and other parameters used in the data reduction. The results obtained from the 180°C data agree very well with those given above. The molecular mechanics calculations yield information consistent with the experimental results.     

The effect of frequency and power density on the ultrasonically-enhanced killing of biofilm-sequestered Escherichia coli

Infection on implanted medical devices is a critical concern because the bacteria are recalcitrant to antibiotic therapy; currently the only way to eliminate the infection is to remove the device. We have found that low-frequency ultrasound renders bacteria more susceptible to antibiotics. The effect of low-intensity ultrasound on the enhancement of antibiotic action against biofilm bacteria was measured by subjecting thick E. coli biofilms for 2 h at 37°C to one of four conditions: (1) incubation in nutrient broth; (2) incubation in nutrient broth with antibiotic; (3) ultrasonication in nutrient broth without antibiotic; and (4) ultrasonication in nutrient broth with antibiotic. Two frequencies (70 and 500 kHz) and several ultrasonic intensities were examined, ranging from 2 to 200 mW/cm². It was determined that low-intensity ultrasound significantly enhanced killing of biofilm E. coli by gentamicin. This enhancement increased with increasing ultrasonic intensity and decreased with increasing frequency. A mathematical model of ultrasonically-enhanced transport in cylindrical pores and channels shows that gentamicin transport increases with ultrasonic intensity and decreases with increasing frequency. However, the magnitude of increased transport is so small that it is difficult to attribute enhanced killing of bacteria to enhanced antibiotic transport through the pores and channels of the biofilm; therefore, other mechanisms must play a role. The use of low-intensity ultrasound in conjunction with antibiotic treatment may prove to be a viable clinical method of eliminating biofilm infections from the surfaces of implanted medical devices.     

Yeast cells long-term interaction with asbestos fibers

Saccharomyces cerevisiae was supported on chrysotile, crocidolite and lixiviated chrysotile. Samples of the supported cells and free cells were observed by confocal laser scanning microscopy. After 30 days, the free cells showed no viability when stored at 30 °C, and a viability of 40% when stored at 4 °C. Supported cells stored at 30 °C were more viable than the free cells at early times, but showed no viability after 30 days. Samples stored at 4 °C showed that the adhered cells are more viable than the free cells, up to 30 days. Cells supported on chrysotile and lixiviated chrysotile had 80% viability, and on crocidolite 70% viability. Scanning electron microscopy showed that cells supported on lixiviated chrysotile are fully covered by the support, but crocidolite fibers adhere less, since they are stiffer. Fermentation experiments performed after 3 years storage showed that four from the five lixiviated chrysotile samples and one of the three crocidolite samples were active. In all cases, a delay time for the onset of fermentation was observed indicating a state of latency.     

Time-dependent fluctuations in infrared vsCH2 frequencies in acyl chain membranes of Acholeplasma laidlawii cells

We measured FT-IR spectra of intact Acholeplasma laidlawii cells grown at 37 °C on palmitic acid (C16:0) or on binary palmitic acid-d31/oleic acid (C16:0-d31/C18:1(9)) at an initial mole ratio of 2:3, which have been previously reported to produce significant fluctuations in CH₂ symmetric stretching (ν_sCH₂) and CD₂ asymmetric stretching (ν_aCD₂) frequencies (Biochim. Biophys. Acta 1279 (1996) 49). Time courses for acyl chain ν_sCH₂ and ν_aCD₂ frequencies determined from fourth derivative spectra are presented. Fluctuations were detected with the C16:0 enriched cells at temperatures above 40 °C as well as with the cells enriched in 2:3 C16:0-d₃₁/C18:1(9). These observations at temperatures above 40 °C for the C16:0 enriched cells were not in agreement with the conclusion in the previous work by Moore et al. Our results have suggested that the 2850 cm⁻¹ ν_sCH₂ band comprises two components arising from trans and gauche conformations, and that the fluctuations in ν_sCH₂ frequency are caused by random temporal changes in the relative intensities of these two components.     

Molecular structure and conformational stability of allylisocyanate—post-Hartree–Fock and density functional theory studies

The molecular structure and conformational stability of allylisocyanate (CH₂CHCH₂NCO) molecule was studied using the ab initio and DFT methods. The geometries of possible conformers, C-gauche (δ=120°, θ=0°) (δ=C=C–C–N and θ=C–C–N=C) and C-cis N-trans (δ=0° and θ=180°) were optimized employing HF/6-31G^*, MP2/6-31G^* levels of theory of ab initio and BLYP, B3LYP, BPW91 and B3PW91 methods of DFT implementing the atomic basis set 6-311+G(d,p). The structural and physical parameters of the above conformers were discussed with the experimental and theoretical values of the related molecules, methylisocyanate and 3-fluoropropene. It has been found that the N=C=O bond angle is not linear as the experimental result for both the conformers and the theoretical bond angle is 173°. The rotational potential energy surfaces have been performed at the HF/6-31G^*, and MP2/6-31G^* levels of theory. The Fourier decomposition potentials were analysed at the HF/6-31G^*, and MP2/6-31G^* levels of theory. The HF/6-31G^* level of theory predicted that the C-gauche conformer is more stable than the C-cis N-trans conformer by 0.41 kJ/mol, but the MP2 and DFT methods predicted the C-cis N-trans conformer is found to be more stable than the C-gauche conformer. The calculated chemical hardness value at the HF/6-31G^* level of theory predicted the C-cis N-trans form is more stable than C-gauche form, whereas the chemical hardness value at the MP2/6-31G^* level of theory favours the slight preference towards the C-gauge conformer.     

Microbiological methods for testing disinfectant efficiency on Pseudomonas biofilm

Biofilms of the Gram-negative bacteria Pseudomonas aeruginosa and Pseudomonas fragi were grown on stainless steel surfaces (AISI 304, 2B) for 4 days in slime broth. These biofilms were treated with four commercial disinfectants. The disinfectants were alcohol-based, tenside-based, peroxide-based and chlorine-based products, covering most disinfectant types used in the food industry. The effects of the disinfectants on the bacterial cells were first investigated in suspension using the permeabilisation test, which is based on fluorescence assessment of hydrophobic 1-N-phenyl-naphtylamine (NPN). The surfaces covered with disinfectant-treated biofilms were investigated using conventional cultivation, impedimetry and epifluorescence microscopy in combination with image analysis of preparations stained with the DNA-stain acridine orange and with the metabolic indicator system CTC-DAPI. The results showed that the tenside-based and peroxide-based disinfectants permeabilised the cells in suspension. The overall biofilm results showed that of the agents tested, the peroxide-based and chlorine-based disinfectants acted most effectively on cells in biofilms.     

Electron diffraction study of the molecular structure and conformation of gaseous 2-furoyl chloride

The molecular structure of 2-furoyl chloride has been investigated by gas-phase electron diffraction at 86°C. Two distinct conformers were identified, a more stable planar form with the furan oxygen and the carbonyl oxygen syn and a less stable planar (or nearly planar) anti form. Assuming that the two forms differ in their geometries only in the O=C---C---O torsion angles and assuming the furan ring to have C_2v symmetry, the results for some of the distances (r_a) and angles (_a) are: r(C---H) = 1.110(20) Å, r(C=O) = 1.207(6) Å, r(C---O) = 1.378(10) Å, r(C??? = 1.465(13) A, (r(C---C)) (average carbon—carbon distance in the furan ring) = 1.392(8) A Δr(C---C) (difference between single and double carbon—carbon distances in the furan ring) = 0.069 A (assumed), r(C---Cl) = 1.787(6) A, C=C---COCl = 131.6(9)°, C=C---O = 110.9(4)°, C=C---H = 127.7(13.4)°, C---C=O = 125.8(8)° and C---C---Cl = 111.8(6)°. At 359 K the observed amount of the conformer with the oxygen atoms syn was 69.8(14.2)%.     

The molecular structure and conformational composition of epichlorohydrin as determined by gas phase electron diffraction

The molecular structure of gaseous epichlorohydrin has been investigated using electron diffraction data obtained at 67°C. The conformational composition at this temperature is such that the molecules exist predominantly in a gauche-2 conformer (where the C---Cl bond is 160° away from the C---O) bond). Refinements showed that 33% (σ = 4) of the molecule exist in the gauche-1 form. The important distances (r_g) and angle () with the associated uncertainties are r(C---H) = 1.095(5) Å, r(C---O) = 1.442(3) Å, r(C---C) = 1.475(8) Å, r(C---C_M) = 1.523(7) Å, r(C---Cl) = 1.788(2) Å, CCO = 114° (1), CCC_M = 119°(1), ClCC = 108.9° (7), and Tau(ClCCO) = −150°(10) (gauche-2) and Tau(ClCCO) = 78° (10) (gauche-1).     

Stainless steel modified with poly(ethylene glycol) can prevent protein adsorption but not bacterial adhesion

The surface of AISI 316 grade stainless steel (SS) was modified with a layer of poly(ethylene glycol) (PEG) (molecular weight 5000) with the aim of preventing protein adsorption and bacterial adhesion. Model SS substrates were first modified to introduce a very high density of reactive amine groups by the adsorption of branched poly(ethylenimine) (PEI) from water. Methoxy-terminated aldehyde-poly(ethylene glycol) (M-PEG-CHO) was then grafted onto the PEI layers using reductive amination at the lower critical solution temperature (LCST) of the PEG in order to optimize the graft density of the linear PEG chains. The chemical composition and uniformity of the surfaces were determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) in the imaging mode. The effects of PEI concentration and different substrate pre-cleaning methods on the structure and stability of the final PEG layer was examined. Piranha solution proved to be the most effective method for removing adventitious hydrocarbon contamination, compared to cleaning with ultrasonication in organic solvents, and was the SS substrate that produced the most stable and thickest PEI layer. The surface density of PEI was shown to increase with increasing PEI concentration (up to 30 mg/ml), as determined from XPS measurements, and subsequently produced the PEG layer with the highest density of attached chains. In model experiments using β-lactoglobulin no protein adsorption was detected on the optimized PEG surface as determined by XPS and ToF-SSIMS analysis. However, neither the adhesion of a Gram-negative (Pseudomonas sp.) nor a Gram-positive (Listeria monocytogenes) bacterium was affected by the coating as equal numbers adhered to all surfaces tested. Our results show that preventing protein adsorption is not a prerequisite stopping bacterial adhesion, and that other mechanisms most likely play a role.     

Sub-ambient temperature effects on the separation of monosaccharides by high-performance anion-exchange chromatography with pulse amperometric detection: Application to marine chemistry

The effects of column temperature in the range 10–45°C using high-performance anion-exchange chromatography (HPAEC) and pulse amperometric detection are described for the determination of monosaccharides. The influence of temperature was tested with an isocratic elution of NaOH at concentrations varying from 2.5 to 20 mM and with a post-column addition of 1 M NaOH. The results showed that small changes of temperature greatly affect retention times and resolution (R_s) of monosaccharides and particularly those of the both pairs xylose–mannose and rhamnose–arabinose which cannot be simultaneously detected at usual room temperature (25°C). Our results suggest that a subambient temperature of 17°C and an eluent concentration of 19 mM are the more appropriate conditions for an acceptable separation (R_s rha/ara=1.02, R_s man/xyl=0.70) in a short analytical run time (35 min). The results showed that within the range of temperatures studied, enthalpy and entropy are invariant of temperature indicating that changes in the retention processes are mainly due to temperature than other associated changes in the system. This study demonstrated the importance of controlling temperature during HPAEC of monosaccharides, both to accomplish highly reproducible retention times and to achieve optimal separation of sugars. This method gave acceptable results for detection of marine sugars.     

Characterization of bovine serum albumin adsorption on chromium and AISI 304 stainless steel, consequences for the Pseudomonas fragi K1 adhesion

Adsorption of BSA on the surface of chromium and 304 stainless steel, has been characterized by Contact Angle Measurements, X-ray Photoelectron Spectroscopy (XPS) and Infrared Reflection Absorption Spectroscopy (IRRAS). Bacterial adhesion has been tested and compared on these two materials before and after pre-conditioning the surface with BSA. Chromium and stainless steel surfaces, when covered by a natural oxide layer, exhibit different energetic characteristics as shown by their γ_s^- and γ_s^LW respective values. These data vary upon immersion in BSA solutions, tending towards common values for duration of immersions. After immersion in BSA solutions, the evolution of the N 1s XPS signal, specific for the BSA, suggests that the surface is nearly saturated in a few minutes. Longer times of immersion only lead to a re-ordering of the adsorbed layer. Immersion tests in dilute BSA solutions (0.01 g/l) enabled us to make clear a higher reactivity of chromium towards the protein compared to stainless steel. These differences are cancelled at higher BSA concentrations (1 g/l). IRRAS spectra of BSA adsorbed on the two substrates demonstrated the appearance of amide I and amide II bands with small shifts and intensity variations supporting orientation changes of the protein when the concentration or immersion time varies. A model for the building up of the BSA layer is proposed, which accounts for these data. Chromium and stainless steel surfaces, also have different behaviours towards adhesion of Pseudomonas fragi K1, whereas surfaces that are pre-conditioned by BSA behave in a similar way. The overall number of adherent bacteria is decreased on stainless steel, whereas it is hardly affected on chromium. On both surfaces, the fraction of viable cells is increased.     

Sunna 535-nm photo-fluorescent film dosimeter response to different environmental conditions

Evaluations on the influence of environmental variabilities on the red fluorescence component of the Sunna Model γ photo-fluorescent dosimeter^TM have previously been reported. This present paper describes the environmental effects on the response of the green fluorescence component of the same dosimeter, which is manufactured using the injection molding technique. The results presented include temperature, relative humidity, and light influences both during and after irradiation. The green fluorescence signal shows a significant dependence on irradiation temperature below room temperature at 1%/°C. Above room temperature (approximately 24–60°C), the irradiation temperature effect varies from −0.1%/°C to 1.0%/°C, depending on the absorbed dose level. For facilities with irradiation temperatures between 30°C and 60°C and absorbed dose levels above 10 kGy, irradiation temperature effects are minimal. Light-effects results indicate that the dosimeter is influenced by ultraviolet and blue wavelengths during irradiation as well as during the post-irradiation stabilization period (approximately 22 h), requiring the use of light-tight packaging. Results also show that the dosimeter exhibits negligible effects from ambient moisture during and after irradiation when in the range of 33–95% relative humidity.     

Isothermal kinetic analysis of the thermal decomposition of magnesium hydroxide using thermogravimetric data

In the present study, the kinetics of the thermal decomposition of magnesium hydroxide is investigated, using isothermal methods of kinetic analysis. For this purpose, experiments in thermogravimetric analyser were carried out in standard values of temperature (350°, 400°, 450° and 500°C) which resulted in weight loss percent as a function of time. The data were further modified to give fraction reacted ‘' versus time to be tested in various forms of ‘' functions. In order to determine the mechanism of the magnesium hydroxide decomposition and the form of the conversion function which governs the dehydroxylation of Mg(OH)₂, four different methods of isothermal kinetic analysis were used. Applying each of these methods to the data, it was concluded that the nucleation mechanism predominates the Mg(OH)₂, decomposition for all values of temperature tested; at 350°C the kinetic model which represents the experimental data is that of reaction at phase boundaries (random nucleation), F₁: ln(1−)=kt) while for the higher temperatures 400°, 450° and 500°C the kinetic equation of nucleation and development in two dimensions, A₂: [−ln (1−)]^1/2=kt was found to fit better the experimental results. The activation energy was evaluated applying two alternative methods; the Arrhenius plot, using maximum rates of reaction, from which the activation energy was evaluated to be 20.54 kcal/mol. An alternative method based on plots of ln t versus 1/T corresponding to the same value of ‘' gave values of 10.72, 13.82 and 16.31 kcal/mol for ‘' values of 0.25, 0.50 and 0.75, respectively.