Freeze-dried Material Research Articles

Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders.

To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). Spray-dried powders with a moisture level (approximately 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.

Purification of Fumonisin B2 Isolated from Rice Culture

Procedures are presented for the production, isolation, and purification of the mycotoxin fumonisin B2 from cultures of Fusarium moniliforme MRC 826 grown on rice. Fumonisins B1, B2, and B3 were extracted with acetonitrile:water, and the extracts were filtered and reduced in volume with a rotary evaporator. Fumonisin B2 was isolated from the other fumonisins B1 and B3 by preparative reversed-phase liquid chromatography (LC). Fractions that contained fumonisin B2 were concentrated to remove acetonitrile, and the remaining water fraction was frozen and freeze dried. The freeze-dried material was dissolved in a minimum amount of methanol and separated by preparative, centrifugally accelerated, radial, thin-layer chromatography on a silica gel-coated plate. An applicator that precisely controlled the rate of application of extract to the sorbent material was used to minimize the width of the band of extract. Fractions were eluted from the spinning plate with a linear gradient of (A) chloroform:acetone (4:3) a...

Effects of a homoanatoxin-a-containing extract from oscillatoria formosa (cyanophyceae/cyanobacteria) on neuromuscular transmission

Experimental investigations were carried out with cultured and lyophilized material of the toxigenic strain Oscillatoria NIVA-CYA 92. This organism is classified as Phormidium formosum (Boryex Gom.) Anagnet kom. Aqueous extracts of the algal material, containing the bioactive secondary amine alkaloid 2-(propan-l-oxo-l-yl)-9-azabicyclo(4,2,1)non-2ene (homoanatoxin-a) in an amount of 2.57 μg/mg lyophilized material, were tested for acute in vivo toxicity in mice, and for toxicity on neuromuscular transmission by means of electrophysiological methods on the isolated phrenic-nerve hemidiaphragm from rat and in the frog rectus abdominis assay. Acute toxic effects in mice were observed by i.p. and oral (by gavage) administration. Lethal doses were in the range 112–225 and 1125–2250 mg of freeze-dried algal material per kg body weight (i.e. 288–578 and 2890–5780 μg homoanatoxin-a/ kg body weight), respectively. The nerve-initiated muscle contractions in the rat diaphragm were blocked by about 0.125 mg cyanophyte material per ml bath solution (i.e. 0.32 μg homoanatoxin-a/ml or 1.8 μM), but muscle contractions, although slightly reduced, could still be elicited by direct electrical stimulation of the muscle. The compound action potentials recorded from the main phrenic-nerve trunk were not affected. An additive blocking effect on partly curarized preparations was observed and cholinesterase inhibition by physostigmine (eserine) transiently augmented the muscle twitch contraction in preparations partly blocked by the extract. Intracellular recordings from single muscle fibres of homoanatoxin-a-treated rat hemidiaphragm disclosed a partial depolarization and a decrease in the endplate potential to subthreshold level simultaneously with a decrease and then complete disappearance of the miniature endplate potentials. The neuromuscular transmission block was reversed by washing. The extract produced muscle contractions in the frog rectus abdominis assay. Homoanatoxin-a in the algal material was readily absorbed from the gastrointestinal tract in mice. Blockade of the neuromuscular transmission of the respiratory muscle may partly explain the acute toxic effects observed in mice. Thus, the main target of the homoanatoxin-a action at the mammalian neuromuscular junction was traced to the postsynaptic nicotinic acetylcholine receptor channel complex, where it reduced the sensitivity to the transmitter substance.

Characterization of the glycation of albumin in freeze-dried and frozen human serum.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

Effect of Drying Temperature on the Stability of Polyphenols and Antioxidant Activity of Red Grape Pomace Peels

The effect of drying temperature (60, 100, and 140 °C) on the polyphenols' content and antioxidant activity of red grape pomace peels was studied. Freeze-dried samples were used as reference. Differences on the CIE-LAB color, total extractable polyphenols, condensed tannins, UV−vis spectra, and antioxidant activity were evaluated. When drying temperature was 100 and 140 °C, a significant reduction in both total extractable polyphenols (18.6 and 32.6%) and condensed tannins (11.1 and 16.6%) was observed, as well as a decrease of 28 and 50% in the antioxidant activity of the samples, respectively. Hue angle and total color difference in the sample dried at 140 °C were significantly higher than in the freeze-dried reference material. A red color loss at 140 °C was also confirmed by lower absorbance values in the spectra at 525 nm. Drying at 60 °C did not significantly affect the sample characteristics evaluated. Keywords: Wine byproducts; grape pomace peels; antioxidant activity; drying temperature

Frozen state transitions in relation to freeze drying

Freeze-drying is used as a gentle dehydration method for heat sensitive materials especially in food and pharmaceutical industries. Most materials, including dissolved sugars in water, do not crystallise during freezing prior to freeze-drying. Supersaturated, freeze-concentrated solutions are amorphous materials and they solidify into a glassy state when their temperature is depressed to below the glass transition temperature,T g. Differential scanning calorimetry has been used to show that maximally freeze-concentrated sugar solutions, when properly frozen, show during heating a glass transition,T g ′ , which is followed by ice melting endotherm with onset atT m ′ . Low molecular weight materials are difficult to freeze-dry as they have lowT g ′ , andT m ′ slightly above T g ′ . High molecular weight materials, such as carbohydrate polymers, exhibit improved dehydration characteristics and they have g ′ and m ′ at about the same temperature close to the melting point of pure water. The amorphous, glassy structure typical of freeze-dried materials is formed during prefreezing and retained after removal of ice and the unfrozen water from the freeze-concentrated material. Dehydration temperatures belowT g ′ allow removal of ice within the solid, glassy solutes, but temperatures aboveT m ′ result in collapse. The frozen state transitions and properties of freeze-dried materials can be shown in state diagrams which are used to derive proper freeze-drying conditions and storage requirements for various materials.

Cell lysis and release of particulate polysaccharides in extensive marine mucilage assessed by lipid biomarkers and molecular probes

During the massive mucllage event in the northern Adriatic Sea in July 1991 samples of macroaggregate were fixed in different ways: with formaldehyde, deep frozen and freeze-dried. Conventional microscopy (light and epifluorescence) revealed different autotrophic species embedded in gelatinous matl-ix. Cyanobacteria and heterotrophic bacteria were also identified. Scanning confocal laser microscopy (SCLMI and fluorescent molec~~ la r p obes (the lectins concanavalin A and UEA-I) showed wall-free cytoplasm and particulate polysacchar~des leaklng from the envelopes of broken cells in the matrix. The extensive cell lysis was supported by the observation of cytoplasn1-free cytoskeletons, stained by the molecular probe phalloidin High concentrations of triglycerides (30Y0 of total lipids) and free fatty aclds (22'2%) along with very low concentrations of phospholipids ( 2 % ) also indicated massive cell degradation in freeze-dried material. The mucllage observations were compared with those of a natural plankton community grown under hlgh nutrlent conditions using the same techniques. Free polysaccharides were observed as globular flocs (marine snow) during in situ enrichment experiments and inti-acellular polysaccharides as carbon storage materials In autotrophic organisms. No strings, filaments, layers, cell lysis or lipid classes indicating strong cell biodeterioration were observed in a 1 mo controlled experiment during an algal bloom.

Determination of Copper, Cadmium and Lead in Biological Samples by Electrothermal Vaporization Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

Electrothermal vaporization isotope dilution inductively coupled plasma mass spectrometry (ETV-ID-ICP-MS) was applied to the determination of Cu, Cd and Pb in several biological samples. The isotope ratios for each element in each analytical run were calculated from the peak areas of each isotope. Various chemical modifiers were tested to obtain the optimum signal of cadmium and palladium was selected. The pyrolytic graphite platforms of the graphite tubes were then pretreated with palladium by three different methods, thermal pretreatment, electroplating and sputtering, and after careful evaluation of the analytical performance of these methods, thermal pretreatment of the platform with Pd before each analysis was selected. The ETV-ID-ICP-MS method was applied to the determination of Cu, Cd and Pb in dogfish liver reference material DOLT-1, dogfish muscle reference material DORM-1 and freeze-dried urine standard reference material NIST SRM 2670. The results agreed satisfactorily with the certified values.

Characterization of highly polymerized procyanidins in cider apple ( Malus sylvestris var. kermerrien) skin and pulp

Freeze-dried materials of cider apple pulp and skin were submitted to three successive solid-liquid extractions. The water-acetone extracts contained significant amounts of condensed tannins; thioacidolysis revealed that they were a mixture of highly polymerized procyanidins mainly constituted of (−)-epicatechin units. Electrospray ionization mass spectrometry showed a complete series of polymeric procyanidins with a degree of polymerization up to 17.

Effect of particle size on in vitro fermentation of silages differing in dry matter content

The objectives of this study were a) to examine the effect of particle size and silage dry matter (DM) content on the rate and pattern of fermentation of fresh silages in vitro as an aid to modelling the in vivo situation and b) to compare the rate and pattern of fermentation of fresh silage samples with those obtained for freeze-dried material.

Moisture-Dependent Crystallization of Amorphous Lamotrigine Mesylate

A commercially available computer-controlled vacuum moisture balance was used for determining moisture sorption isotherms of freeze-dried and spray-dried lamotrigine mesylate drug substance and freeze dried drug product containing mannitol. The presence or absence of desorption hysteresis and the characteristics of the weight-versus-time profile as a sample was exposed to a defined relative humidity ramp were sensitive indicators of moisture-induced crystallization. Combination of the moisture sorption data with polarized light microscopy, differential scanning calorimetry, and X-ray powder diffraction provided qualitative verification of the crystallization with <50mg of sample. The normalized water loss during crystallization was used to detect as little as 2% amorphous content in physical mixtures of amorphous and crystalline lamotrigine mesylate. Moisture sorption, water plasticization, and crystallization properties of amorphous forms prepared by spray drying and freeze drying were nearly identical. Cofreeze-drying lamotrigine mesylate with D-mannitol resulted in a mixture of amorphous lamotrigine mesylate with properties similar to those of spray-dried or freeze-dried materials and crystalline D-mannitol. The amount of water needed for crystallization over a time scale observable in the moisture balance was considerably more than the amount needed to lower the glass transition temperature of the sample to the operating temperature of the instrument. This result illustrated the importance of time scale effects in determining critical moisture levels for crystallization from the amorphous state.

Infraspecific taxonomy and essential oil chemotypes in sweet basil, Ocimum basilicum.

Essential oil compositions of fresh and freeze-dried leaves were determined for 16 accessions of Ocimum basilicum belonging to different varieties to see whether they could be used as infraspecific taxonomic characters. One accession of O. x citriodorum was also studied. Some 30 monoterpenoids, sesquiterpenoids and phenylpropanoids were identified, the major components (more than 20% of the total essential oil composition in one or more accessions) being geranial and neral in O. x citriodorum, and linalool, methyl chavicol, eugenol, methyl eugenol and geraniol in O. basilicum. Based on a combination of the latter compounds, five major essential oil profiles could be distinguished in the accessions studied for O. basilicum. These profiles were largely the same for fresh and freeze-dried material of the same plant, although in the dried leaves, methyl chavicol and eugenol concentrations had generally declined in comparison to those of linalool. There appeared to be little correlation between essential oil patterns and varietal classification within O. basilicum. In view of the chemical heterogeneity of O. basilicum and its use as an essential oil-producing crop, culinary herb, medicinal plant and insect-controlling agent, in all of which chemicals play an important role, the infraspecific classification of this taxon should take chemical characters into consideration. A system for the classification of essential oil chemotypes in O. basilicum is proposed.

Validation and quality control of methylmercury determinations by means of capillary electrophoresis

Methyl-Hg is one of the most dangerous pollutants in the environment. It is highly toxic and accumulates through the food chain. Tuna fish especially tends to concentrate Hg in the form of methyl-Hg. Quality assurance is a well established routine in analytical laboratories to control the variability of analytical results and to maintain adequate stability, detection power and reproducibility. Capillary electrophoresis (CE) has shown in recent years powerful capabilities for the resolution and analysis of very complex samples. The high resolution and peak capacity characteristics of this technique could be used favourably in speciation studies of organo-Hg compounds. Since the time needed for separation is usually very short in this technique, it lends itself to routine analysis. The application of quality control approaches in the analysis of methyl-Hg in tuna freeze-dried materials using CE is reported.

Influence of drying method and ageing on chemical and physical properties andin vitrodegradation characteristics of grass and maize samples

SUMMARYThe influence of different drying conditions on the chemical composition, physical properties,in vitroorganic matter degradability and fermentation kinetics of forages was investigated using young and old grass (Lolium perenne) samples (harvested on 15 June and 9 July 1992 at Lelystad, The Netherlands) and young and old maize (Zea mayscv. Scana) stem samples (harvested on 19 August and 30 September 1991 at Lelystad). The samples were either freeze-dried with a maximum sample temperature of 10 °C, dried in a vacuum at 20 °C or air-dried at 30, 50, 70 and 105 °C. The different drying methods had little effect on ash, acid detergent fibre (ADF), acid detergent lignin (ADL), crude fibre and crude protein (CP) contents andin vitrodegradation of the forage samples. However, some effects were found for sugars and phenolic acids. The neutral detergent fibre (NDF) content in protein-rich samples and the fermentation kinetics in rumen fluid differed significantly according to drying method. In samples dried other than by freeze-drying, proteins were bound to the NDF content and in some cases an effect on the amount of soluble sugars was also seen. Physical properties of the samples were determined by differential scanning calorimetry (DSC) and differences were found between freeze-dried materials and those dried at 70 °C. The influence of age on the maize samples was very pronounced, whereas it had little effect on the characteristics of the grass samples, with the exception of a decreased CP content and an increased sugar content after acid hydrolysis.

Trace elements in a commercial freeze-dried human urine reference material.

A large batch of freeze-dried human urine reference material, Seronorm Trace Elements Urine, Lot 101021, was prepared commercially (Nycomed Pharma AS, Oslo, Norway) for quality control purposes in trace element analysis. Analytes are being determined by a voluntary, international co-operative effort so that the material will be available to the scientific community at modest cost. The material is in stoppered glass vials and is to be reconstituted with 5.00 ml of water prior to use. We have evaluated the trace element content for several elements, including chromium and zinc, elements for which we have two independent methods available for the determinations, namely isotope dilution mass spectrometry (IDMS) and atomic absorption spectrometry (AAS). We also report on other trace elements measured by IDMS alone, such as Se, for which we have enriched stable isotopes available. Results for chromium indicate a mean +/- standard deviation (n = 10) of 1.2 +/- 0.3 (by IDMS) and 1.4 +/- 0.3 (by AAS) ng Cr per ml of reconstituted urine, indicating possible inhomogeneity and/or contamination (21-25% relative standard deviation, RSD). Approximately half of the observed chromium originates from the sample container. The values observed for zinc were 590 +/- 90 ng ml-1 (15% RSD) for freshly reconstituted material, 760 +/- 60 ng ml-1 (8% RSD) for material reconstituted 4 d earlier, and 940 +/- 60 ng ml-1 (6% RSD) 2 months after reconstitution. Selenium values by IDMS were very reproducible, with a mean concentration of 16 +/- 0.15 ng g-1 (0.9% RSD), suggesting little or no contamination and a high degree of sample homogeneity for this element. The source of potential contaminants has been evaluated by multielement determinations of leachates of the vials and stoppers. Elements noted in significant amounts include B, Ba, Sr, Mo, Cu and Zn, with most of the zinc coming from the rubber stopper.

The physical state of nafcillin sodium in frozen aqueous solutions and freeze-dried powders.

The purpose of this study was to develop a better understanding of the physical chemistry of freeze drying of lyotropic liquid crystals using nafcillin sodium as a model solute. Solutions and freeze-dried powders of nafcillin sodium were studied by polarized light microscopy, differential scanning calorimetry, x-ray powder diffraction, and water vapor adsorption. Differential scanning calorimetry thermograms of nafcillin sodium solutions contain a melting endotherm at approximately -5.5 degrees C and, depending on the concentration and heating rate, a crystallization exotherm immediately after this endotherm followed by the melting endotherm of ice. When the sample is annealed at -4 degrees C, both the endotherm and exotherm are eliminated, and a new endotherm appears at approximately -1 degree C on the shoulder of the ice-melting endotherm. The data are interpreted as melting of a liquid crystalline phase, followed by crystallization. X-ray powder diffractograms of unannealed freeze-dried nafcillin sodium are consistent with a lamellar liquid crystal. Diffractograms of annealed freeze-dried nafcillin sodium indicate crystalline material which is a different crystal form than the monohydrate starting material. Moisture adsorption isotherms of the freeze-dried annealed (crystalline) and unannealed (liquid crystalline) nafcillin sodium show different affinities for moisture compared to the crystalline starting material. Solid-state stability data demonstrate that the freeze-dried liquid crystalline form of nafcillin sodium is much less stable than the freeze-dried crystal-line material. The literature recognizes two types of solute behavior on freezing, where the solute either crystallizes from the freeze concentrate or remains amorphous. Lyotropic liquid crystal formation during freezing represents a separate category of freezing behavior, the physical chemistry of which is worthy of further investigation.

Pilot study of labelling bovine muscle tissue with 14C

A pilot study has been performed to assess the feasibility of labelling bovine muscle tissue with 14C, in order to produce material suitable for intercomparison exercises. Approximately 1 kg of tissue was prepared by grinding, blending, labelling with 14C-methylated bovine haemoglobin and thoroughly mixing by repeated blending. The product was sealed in cans and sterilized to preserve it. Portions of raw material were dried to test the homogeneity of moisture content. Homogeneity of 14C in the natural and labelled materials was tested by analyzing sub-samples of 2–3 g in weight of the freeze-dried materials. The 14C content was determined by combusting and collecting the CO 2 for liquid scintillation counting. The material was found to be homogeneous to the extent of 2–4% (R.S.D.), which is comparable with the R.S.D. of the 14C measurements. The 14C concentration in the labelled material was 8190 ± 220 Bq·kg −1 of carbon. It was concluded that bovine muscle prepared in this manner would be suitable for intercomparison exercises. The 14C level in unlabelled tissue was found to be 260 ± 7 Bq·kg −1 of carbon, which is similar to the level of 14C previously determined in milk and both are consistent with levels measured in vegetation in Eastern Ontario.

Purification of ptaquiloside, a carcinogen from Pteridium aquilinum

Ptaquiloside, the major carcinogen of bracken fern ( Pteridium aquilinum), was isolated in high yield from milled freeze-dried plant material. Because the compound is unstable, a new method was devised to avoid steps which caused loss of activity. Modern multipulse and 2D NMR techniques allowed confirmation of the previously proposed structure, although some assignments were shown to be incorrect.

Effect of Plant Maturity and Preservation Method on In Vitro Protein Degradation of Forages

The influence of maturity and method of conservation on protein degradation was determined for four different forage species. Alfalfa, smooth bromegrass, and reed canarygrass were harvested at three maturities, and whole plant corn was harvested at two maturities. Samples of each forage were freeze-dried or wilted and then ensiled in mini silos at two DM contents. Additional samples of all forages except corn were field-dried to hay. Ground sample was incubated for 0, 2, and 24h with crude enzyme extract from ruminal contents. Degraded protein as a percentage of total CP was determined as the amount of protein that was soluble in TCA (80g/L) after degradation. Increased maturity resulted in lower protein degradation for alfalfa, bromegrass, and canarygrass. For example, the most mature alfalfa or bromegrass, respectively, had 77 or 63% as much N that was soluble in TCA after 2h of incubation with ruminal enzyme than the least mature forage of the same species. Although protein degradation was higher for ensiled than for dried forage, silage DM content had no consistent effect. Freeze-dried material generally had less degraded protein than hay, but protein degradation of bromegrass at 24h was lower for hay than for freeze-dried samples. Protein degradation of forages was highly variable and depended on plant maturity and conservation method.

Preparation and homogeneity study of a mussel candidate reference material for certification of organotin compounds

Organotin compounds are currently determined in various environmental matrices (in particular water, sediment and shellfish) to form the basis of control measures for the levels of contamination, as required in some EC Directives. Accurate measurements are a prerequisite for the enforcement of this legislation, which implies that good quality control of determinations should be ensured. One of the tools for achieving accuracy is to use certified reference materials (CRMs). With regard to tin species, however, CRMs are rather scarce and mainly concern sediment and fish matrices certified for their butyltin content. The present study deals with the preparation of a mussel candidate reference material and the verification of its homogeneity prior to the certification of the contents of butyl- and phenyl-tin compounds. 100kg of mussels (Mytilus edulis) were collected, carefully washed with fresh water in order to eliminate matrix salts and immediately frozen by immersion in liquid nitrogen. The mussels were shelled (in the frozen state) and the edible part was stored in thermally sealed polyethylene bags which were stored at −25 °C. The frozen material was ground in a Teflon-coated grinding mill, spread on sterilized flat trays and freeze-dried. The freeze-dried material was then ground for 15 days in a zirconia ball mill and sieved on a titanium sieve to separate the fibrous part. Successively, the material was mixed for 15 days under argon atmosphere in a special polyethylene-lined mixing drum and bottled in brown Pyrex-glass bottles. 21 bottles were selected during the course of the bottling procedure to verify the within- and between-bottle homogeneity of the overall batch. The within-bottle homogeneity was verified by determining the organotin (butyl- and phenyltins) content for subsamples of 500 mg in ten replicates from one bottle, whereas the between-bottle homogeneity was controlled by one organotin measurement from each of 20 bottles. The analyses were performed by GC-MS. The results showed that the homogeneity of the material makes it suitable as a candidate reference material.