Frozen Storage Stability Research Articles

The use of orthogonal analytical approaches to profile lipid nanoparticle physicochemical attributes

Abstract Lipid nanoparticles have become a major disruptor within the drug delivery field of complex RNA molecules. The wide applicability of prototype nanomedicines have the potential to fill clinical requirements against current untreatable diseases. The uptake and implementation of analytical technologies to evaluate these prototype nanomedicines have not experienced similar growth rates hindering the translation of LNPs. Here, we evaluate a model RNA-LNP formulation with a selection of routine, and high-resolution orthogonal analytical techniques across studies on manufacturing process parameter impact and formulation stability evaluation under refrigerated and ultra-low temperatures. We analysed model cationic RNA complexed lipid nanoparticle formulation through process impact on formulation critical quality attributes, short term refrigerated stability evaluation, and frozen storage stability using zetasizer dynamic light scattering and nanoparticle tracking analysis. We also evaluated freeze/thaw induced stress on LNP formulation using high resolution field-flow fractionation. Statistical analysis and correlations between techniques were drawn to further enhance our understanding of LNP formulation design, and physiochemical attributes to facilitate LNP formulation clinical translation.

Effect of combined enzyme and ultrasound treatment on the structure and gel properties of soy protein isolate: A comparative study of alkaline protease and pepsin

This study aimed to research the effects of enzyme and ultrasound combined modification on the structure and gel properties of soy protein isolate (SPI). Different modified protein samples were obtained by hydrolyzing soy isolate with alkaline protease and pepsin at 0%, 0.1%, 0.5%, and 1.0% of hydrolysis by ultrasound treatment (400 W, 20 min). The results showed that the SPI particle size, which modified by combined enzyme and ultrasound treatment (co-modified), was significantly lower (P < 0.05) and the particle size was more evenly distributed compared with those of the single modified samples. The best improvement in solubility and turbidity was observed for the co-modified SPI. The Fourier transform–infrared spectroscopy showed the most significant changes in the secondary structure content of the co-modified SPI. The protein structure was unfolded, and the contents of free sulfhydryl and H0 were improved. Additionally, the co-modified SPI gel had a uniform and compact gel network and higher water-holding capacity (WHC) and gel strength. After frozen storage, the combined enzyme and ultrasound treatment showed better frozen storage stability than single treatment: the WHC of the gel decreased slightly, the gel strength increased slowly, and the gel network was less damaged. In conclusion, combined enzyme and ultrasound treatment could be effective in improving the SPI structure and gel properties.

Effects of different storage temperatures and time on frozen storage stability of steamed bread.

This research intended to explore the effects of different frozen storage temperatures and time on the stability of steamed bread. The quality characteristics, moisture content and microstructure of steamed bread were determined after quick-frozen for 30 min at -32 °C and frozen storage at -6, -12, -18, -24 and -30 °C for 1-4 weeks. When the frozen storage temperature is lower, the moisture content, specific volume, pH and the strong bound water in the steamed bread increase, the water loss rate and the contents of freezable water, the weak bound water and free water decreased. With the extension of frozen storage time, the pH value and water loss of steamed bread first increased and then decreased, while the trend of water content was opposite. The specific volume, cohesion and elasticity of steamed bread decreased, while the freezable water content, hardness and chewiness increased. The bound water of steamed bread gradually migrated to free water. In addition, the longer the frozen storage time and the higher the temperature, and the more serious the damage to the microstructure was. The shelf life of steamed bread frozen storage at -12 °C could be up to 3 weeks, and the quality of steamed bread stored at -30 °C for more than 3 weeks was the best. © 2022 Society of Chemical Industry.

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – part 2: Aggregate formation and oxidation

We examined the impact of monoclonal antibody (mAb) and buffer concentration, mimicking the cryoconcentration found upon freezing in a 2 L bottle, on mAb stability during frozen storage. Upon cryoconcentration, larger protein molecules and small excipient molecules freeze-concentrate differently, resulting in different protein to stabiliser ratios within a container. Understanding the impact of these shifted ratios on protein stability is essential. For two mAbs a set of samples with constant mAb (5 mg/mL) or buffer concentration (medium histidine/adipic acid) was prepared and stored for 6 months at −10 °C. Stability was evaluated via size-exclusion chromatography, flow imaging microscopy, UV/Vis spectroscopy at 350 nm, and protein A chromatography. Dynamic light scattering was used to determine kD values. Soluble aggregate levels were unaffected by mAb concentration, but increased with histidine concentration. No trend in optical density could be identified. In contrast, increasing mAb or buffer concentration facilitated the formation of subvisible particles. A trend towards attractive protein-protein interactions was seen with higher ionic strength. MAb oxidation levels were negatively affected by increasing histidine concentration, but became less with higher mAb concentration. Small changes in mAb and buffer composition had a significant impact on stability during six-month frozen storage. Thus, preventing cryoconcentration effects in larger freezing containers may improve long-term stability.

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at −80 °C, −20 °C, and − 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure Tg′ of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine Tg′ in formulations without glass forming sugars. Storage below Tg′ did not lead to mAb aggregation. Above Tg′, at −20 °C and − 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below Tg′ completely prevents mAb aggregation and particle formation.

Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years

BackgroundThe use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. ObjectivesWe aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. MethodsWe prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at −70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC–tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland–Altman plot, Deming regression). ResultsAt baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. ConclusionsErythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at −70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Evolution of tissue microstructure, protein properties, and oxidative stability of salted bigeye tuna (Thunnus obesus) meat during frozen storage

The characteristics of ice crystals, tissue recoverability, protein properties, and lipid oxidation were investigated to reveal the effects of salting time (0–8 h) on the frozen storage stability of tuna meat. After 2 w of frozen storage, the ice crystals in tuna meat with different salting time varied in morphological properties, evolving from large columns (0 h) to ellipsoid (0.5–3 h) and plate shape (5–8 h). The ice crystals in tuna meat with salting time of 1–3 h were more stable with the extension of frozen storage. The enhanced water-holding capacity, springiness, and cohesiveness were attributed to the modified morphology and stability of ice crystals and tissue recoverability after thawing with the formation of moderate protein-protein interactions under proper salting conditions. The declined oxidative stability of salted tuna meat was likely associated with the loss of redox balance with the extension of salting as indicated by aggravated oxidation of lipid and myoglobin and discoloration. Salting time must be taken into account for the frozen storage stability of salted aquatic products, which could modify the microstructure of both frozen and thawed meat, protein properties, redox status of tissue, and thus quality attributes.

Effect of different extent of protein oxidation on the frozen storage stability of muscle protein in obscure pufferfish (Takifugu obscurus)

The objective of this study was to explore frozen storage stability of muscle protein of the obscure pufferfish using a semi-model system based on light exposure (LE), oxygen concentration (OC), and multiple freeze-thaw cycles (FTCs). The dorsal muscle of fifteen frozen fish was cut into small pieces (2 cm × 1.5 cm × 1 cm) and divided into 4 groups stored in differing conditions: one was stored in light with 0% oxygen (L-0), one in light with 20% oxygen (L-20), one in dark with 0% oxygen (D-0), and one in dark with 20% oxygen (D-20). All were subsequently subjected to five FTCs. Protein oxidation (PO) levels, protein structure, and water states were all assessed. The results showed that both LE and OC could significantly accelerate sulfhydryl and tryptophan loss, in the order of L-20 > L-0 and D-20 > D-0 during FTCs. Changes of protein structure were in line with the extent of PO indicated by surface hydrophobicity and UV absorbance. An increased PO also affected the water states, with higher PO resulting in immobilized water being more tightly trapped within the protein. However, thawing loss was shown to be independent of PO and the structural changes of myofibrillar protein.

Investigating commercially relevant packaging solutions to improve storage stability of mechanically filleted Atlantic mackerel (Scomber scombrus) produced under industrial conditions

This study investigated the efficacy of three commercially relevant packaging methods (vacuum with water glazing VAC-G; vacuum with seawater VAC-S; shatter-layer packaging SL) to improve frozen storage stability of mechanically filleted Atlantic mackerel at − 25 °C, in comparison to water glazing alone (GL) and storage as whole unglazed, block frozen fish. Besides proximate composition and pH of raw material, quality changes were analysed by free fatty acid content (FFA), water holding capacity (WHC), cooking yield, lipid oxidation (lipid hydroperoxides, PV; non-protein bound thiobarbituric acid reactive substances, TBARS) and sensory profiles of cooked samples after 3.5, 8, 10 and 12 months of frozen storage. Vacuum-packaging was effective in mitigating the PV and TBARS as well as rancid odour and flavour. The inclusion of seawater in VAC-S altered the sensory textural attributes of the mackerel fillet to be more juicy, tender and soft and increased the attribute of salty flavour in the sample. SL delayed rancid odour and flavour by 2 months compared to GL. Processing of mackerel under industrial conditions, including filleting, handling, double-freezing and glazing accelerated the formation of FFA as well as losses of WHC and cooking yield in the fillet regardless the packaging methods.

Serum Folate Forms Are Stable during Repeated Analysis in the Presence of Ascorbic Acid and during Frozen Sample Storage.

Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation. Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1-33 weeks) storage time at -70 °C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms. Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (-0.24% to 0.39%; r c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%-6.05%; r c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate (MeFox), -0.22% (-1.90% to 1.49%; r c = 0.986; n = 214) for folic acid, and 1.49% (-2.71% to 5.88%; r c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than ±5% for samples retested after 4 months: 5-methyltetrahydrofolate P trend = 0.0007, folic acid P trend < 0.0001, MeFox P trend = 0.38, and tetrahydrofolate P trend = 0.0256. The mean ± SD tetrahydrofolate spiking recovery was 96.7% ± 9.4% for serum with added AA, but <50% for serum without added AA. We observed ≤10% loss for most serum folate forms during 4 years of storage at -70 °C. Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms.

The effect of clove oil on frozen storage stability and quality of rainbow trout (Oncorhynchus mykiss)

Bu calismada, 6 ay boyunca dondurulmus alabalik filetolarinin kalitesi uzerine dogal bir koruyucu olarak karanfil yaginin (0.5% ve 1%v/w) etkisi degerlendirildi. Vakum paketlenmis ornekler -18oC’de depolandi. Depolama suresince mikrobiyolojik (Toplam anaerob bakteri, psikrofilik bakteri, laktik asit bakterileri ve maya-kuf) ve duyusal (koku, renk, tekstur) parametreler incelendi. Mikrobiyal gelisme kontrol grubunda depolama suresince en yuksekti. Karanfil yagi vakum paket ile birlikte kullanildigi kombinasyonlarda dondurulmus alabalik filetolarinin duyusal kalitesi uzerine olumlu etki gosterdi. Sonuclarimiza gore karanfil yagi, etkili antibakteriyel ve dogal bir koruyucu olarak dondurulmus gokkusagi alabaligi kalitesini artirmak icin vakum paket ile birlikte kullanilabilir.

Effect of clove oil treatment on the frozen storage Indian mackerel (Rastrelliger kanagurta) steaks

The antioxidant effect of clove on the frozen storage stability of fish steak from Indian mackerel ( Rastrelliger kanagurta ) was studied. Fresh mackerel as a raw material had moisture content 73.99 per cent, crude protein 18.16 per cent, crude fat 3.73 per cent and ash 1.71 per cent. The steaks from whole mackerel were treated with Clove oil 0.5, 1.0 and 1.5 ml per lit. of distilled water for 5 minute. All samples were stored at -18 ± 2 o C for the six months storage and experimental analysis. Based on the overall acceptability scores, 0.5 ml clove oil treatment is found to better consumer acceptance than other samples during storage.

Practical Considerations for Determination of Glass Transition Temperature of a Maximally Freeze Concentrated Solution.

Glass transition temperature is a unique thermal characteristic of amorphous systems and is associated with changes in physical properties such as heat capacity, viscosity, electrical resistance, and molecular mobility. Glass transition temperature for amorphous solids is referred as (T g), whereas for maximally freeze concentrated solution, the notation is (T g'). This article is focused on the factors affecting determination of T g' for application to lyophilization process design and frozen storage stability. Also, this review provides a perspective on use of various types of solutes in protein formulation and their effect on T g'. Although various analytical techniques are used for determination of T g' based on the changes in physical properties associated with glass transition, the differential scanning calorimetry (DSC) is the most commonly used technique. In this article, an overview of DSC technique is provided along with brief discussion on the alternate analytical techniques for T g' determination. Additionally, challenges associated with T g' determination, using DSC for protein formulations, are discussed. The purpose of this review is to provide a practical industry perspective on determination of T g' for protein formulations as it relates to design and development of lyophilization process and/or for frozen storage; however, a comprehensive review of glass transition temperature (T g, T g'), in general, is outside the scope of this work.

Study on quality characteristics of camel burger and evaluating its stability during frozen storage

This study investigated the influence of replacing One-humped camel meat instead of cow meat (0, 25, 50, 75 and 100 %) on quality characteristics and frozen storage stability in burgers. For this purpose thiobarbituric acid test (TBA-RS), cooking characteristics, color parameters, texture and sensory properties were studied during 3 months at −18 °C. Significant differences (p 0.05) in various levels of camel meat and over storage term. Moisture retention of burgers increased with increasing of camel meat content. The sensory panel scores for flavor, texture, juiciness and overall acceptability increased but color scores decreased with increasing the level of camel meat. Cooking yield, fat retention and cohesiveness decreased by increasing the storage period.

Influence of cryoprotectant levels on storage stability of surimi from Nemipterus japonicus and quality of surimi-based products.

A study was undertaken with the aim of reducing the concentration of cryoprotectants in surimi without adversely affecting frozen storage stability. Minced meat from a tropical fish, Nemipterus japonicus, was strained, water leached and mixed with different levels of sucrose-sorbitol (1:1) mixture (henceforth called sugar mixture), quick frozen at -35°C and frozen stored at -20°C. The surimi samples were subjected to storage stability studies for a period of 5months. Water leaching resulted in slight absorption of water by meat and reduction in protein, fat and mineral contents. Surimi was found to have moderately white colour. Sensory evaluation studies were conducted on three products, viz., sausage, patty and cake, prepared using surimi containing different concentrations of sugar mixture. Sugar mixture content varying from 0% (control) to 4% in surimi resulted in products that were more acceptable to the taste panelists compared those with 6% and 8% sugar mixture. During frozen storage of surimi pH and total plate count remained nearly steady for all sugar mixture concentrations and throughout the storage period. Moisture content appeared to remain constant during storage, but decreased with increase in sugar concentration. The salt soluble nitrogen content of surimi and gel strength of sausage prepared from it decreased with storage period in all surimi samples, and increased with sugar mixture concentration. Expressible water content of surimi sausage showed an increasing trend with storage period of surimi and a decreasing trend with sugar mixture concentration. Sensory evaluation parameters-elasticity, sweetness and preference-remained more or less steady during storage. However elasticity and sweetness increased and preference decreased with sugar mixture concentration beyond 4%. Elasticity and gel strength of surimi sausage seemed to be much lower for control compared to even the lowest concentration of sugar (2%) used. A concentration of 2 to 4% sucrose-sorbitol mixture is well-accepted by the consumers in products-surimi sausage, patty and cake and at this range of concentration surimi could be well-preserved at -20°C for at least 5months.

Effect of essential oils treatment on the frozen storage stability of chub mackerel fillets

The effect of bay leaf (BLO), thyme (TO), rosemary (RO), black seed (BSO), sage (SO), grape seed (GSO), flaxseed (FSO) and lemon (LO) essential oil from vegetable extracted on lipid oxidation and some other quality parameter of frozen chub mackerel during frozen storage at −20°C were examined over a period of 11 months. Taste, odour, texture and overall acceptability of control samples were given ‘unacceptable’ scores by the sixth month. Based primarily on sensory data, the shelf-lives of frozen chub mackerel were found 6 month for samples treated with oil of TO, RO, BSO, SO and LO and 7 month for samples treated with BLO, GSO and FSO. During the 11-months storing process of chub mackerel, the values of pH, total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA-N) both in control group samples and samples treated with oils did not reach to deterioration levels. Thiobarbituric acid (TBA) and free fatty acid (FFA) values for all treatments remained lower than TBA and FFA values of control samples throughout the 11 month storage period. Particularly, thyme oil treatment is effective in delaying lipid oxidation. Bay leaf, rosemary, sage, lemon, flaxseed and grape seed oils were fallowed.

The Acid and Alkaline Solubilization Process for the Isolation of Muscle Proteins: State of the Art

The acid and alkaline solubilization processes for isolating muscle protein from ground fish raw materials are investigated by scrutinizing the literature. Following an introduction to the processes, with some underlying chemistry, patents related to the acid and alkaline solubilization process are described together with previously patented methods for processing of protein isolates. Focus is then placed on comparing a range of factors important in fish muscle protein isolation between the acid and alkaline solubilization processes, and classic washing-based surimi technology. The factors addressed were: protein yield, gel quality, color, lipid reduction, lipid oxidation, microbial stability, and frozen storage stability. A long series of studies made with different fish/shellfish species have been used for this purpose. Certain results are summarized in table form (protein yields, gel strength, and whiteness), others only in text form. From this part of the review, it is obvious that the acid process often has certain advantages (e.g., protein yield) and the alkaline process other ones (gel strength, whiteness, lipid removal, lipid oxidation, and total microbial count). Thus, the choice of method depends on the application. It is clear that most species respond differently to acid and alkaline solubilization, which is why the two methods are initially compared. In a section about new processing attempts, the use of the acid and alkaline methodology for isolating proteins from whole fish/shellfish and fish by-products is reviewed together with attempts to recover waste water proteins and attempts to modify the process. Tentative uses of the new protein isolates, e.g., as coatings, protein brines, and emulsifiers are finally described together with some conclusions and future opportunities for the acid and alkaline processes.

Effect of some additives on the inhibition of lizardfish trimethylamine‐N‐oxide demethylase and frozen storage stability of minced flesh

SummaryEffects of some additives on the inhibition of trimethylamine‐N‐oxide demethylase (TMAOase) from lizardfish (Saurida tumbil) muscle were investigated. Sodium citrate and pyrophosphate could inhibit TMAOase activity in a concentration‐dependent manner, most likely because of their chelating property. Sodium alginate was the hydrocolloid possessing the inhibitory activity towards TMAOase (P &lt; 0.05). During the storage of lizardfish mince at −20 °C for 24 weeks, the addition of 0.5% sodium alginate and 0.3% pyrophosphate in combination with 4% sucrose and 4% sorbitol as the cryoprotectants resulted in the retarded increase in TMAOase activities with the coincidental lowered formation of dimethylamine and formaldehyde (FA), compared with the control (without additives) (P &lt; 0.05). The loss in solubility of muscle proteins was also impeded with the addition of those compounds, suggesting their role in the inhibition of TMAOase as well as the retardation of protein denaturation induced by FA.

Effect of a flax seed (Linum usitatissimum) soaking treatment on the frozen storage stability of mackerel (Scomber scombrus) fillets

AbstractThe effect of a flax seed (Linum usitatissimum) soaking on the development of rancidity in frozen mackerel (Scomber scombrus) was studied. Fresh mackerel fillets were soaked in an aqueous flax seed extract for 20 min and then kept frozen (−20 °C) for up to 7 months. A parallel experiment with non‐soaked fillets was carried out under the same conditions. The development of rancidity was measured by biochemical (free fatty acids, peroxides, conjugated dienes and trienes, secondary oxidation products, fluorescent and browning compounds and lipoxygenase activity) and sensory (general aspect, odour and colour) analyses. An inhibitory effect of the soaking treatment on rancidity development was observed according to the peroxide content and the formation of fluorescence and browning. A lower lipoxygenase activity was detected at 1 month in the soaked fillets; after this, no differences were obtained between either type of sample, whose activities at month 7 were negligible. According to the sensory analyses, non‐soaked fillets had fair quality at 1 month and were rejectable at 3 months, while the soaked ones were still of good quality at 1 month and rejectable at 5 months. According to the present results, soaking in an aqueous flax seed extract could be useful for inhibiting the development of rancidity in fatty fish fillets. Copyright © 2006 Society of Chemical Industry

Determination of emamectin residues in the tissues of Atlantic salmon (Salmo salar L.) using HPLC with fluorescence detection.

An accurate, reliable, and reproducible assay for the determination of residual concentrations of emamectin B(1a) in muscle, skin, and intact muscle/skin in natural proportions from Atlantic salmon treated with SCH 58854 (emamectin benzoate) is described. The determinative method was developed and validated using fortified control tissues at five levels over a range of 50-800 ng/g as well as tissues containing incurred levels in the same range. Incurred tissues were obtained from a metabolism study of [(3)H]emamectin benzoate in Atlantic salmon. The assay employs processing of a tissue ethyl acetate extract on a propylsulfonic acid solid phase extraction cartridge, followed by derivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. Following separation using reversed phase HPLC, the amount of derivatized emamectin B(1a) is determined by fluorescence detection. The theoretical limits of detection were determined from the analysis of control tissue matrices to be 2.6, 3.3, and 3.8 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. Likewise, the theoretical limits of quantitation (LOQ) were determined to be 6.9, 8.1, and 9.5 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. The lowest fortification level used for method validation was 50 ng/g, which served as the effective LOQ for the method. The overall percent recoveries (+/-% CV) were 94.4 +/- 6.89% (n = 25) for muscle, 88.4 +/- 5.35% (n = 25) for skin, and 88.0 +/- 3.73% for intact muscle/skin (n = 25). Accuracy, precision, linearity, selectivity, and ruggedness were demonstrated. The structure of the final fluorescent derivative of emamectin B(1a) free base was identified by ESI(+)/LC-MS. The frozen storage stability of [(3)H]emamectin B(1a) in tissues with incurred residues was demonstrated for approximately 15 months by radiometric analysis and for an additional approximately 13 months by fluorometric analysis for a total of approximately 28 months.