Production of a Bioemulsifier with Potential Application in the Food Industry

Biosurfactants are of considerable interest due to their biodegradability, low degree of toxicity, and diverse applications. However, the high production costs involved in the acquisition of biosurfactants underscore the need for optimization of the production process to enable viable application on an industrial scale. The aims of the present study were to select a species of Candida that produces a biosurfactant with the greatest emulsifying potential and to investigate the influence of components of the production medium and cultivation conditions. Candida utilis achieved the lowest surface tension (35.53 mN/m), best emulsification index (73 %), and highest yield (12.52 g/l) in a medium containing waste canola frying oil as the carbon source and ammonium nitrate as the nitrogen source. The best combination of medium components and cultivation conditions was 6 % (w/v) glucose, 6 % (w/v) waste canola frying oil, 0.2 % (w/v) ammonium nitrate, 0.3 % (w/v) yeast extract, 150 rpm, 1 % inoculum (w/v), and 88 h of fermentation. The greatest biosurfactant production and the lowest surface tension were achieved in the first 24 h of production, and the maximum biomass production was recorded at 72 h. The biosurfactant produced from C. utilis under the conditions investigated in the present study has a potential to be a bioemulsifier for application in the food industry.     

Enhanced Production of Bacterial Cellulose by Using Gluconacetobacter hansenii NCIM 2529 Strain Under Shaking Conditions

Bacterial cellulose (BC), a biopolymer, due to its unique properties is valuable for production of vital products in food, textile, medicine, and agriculture. In the present study, the optimal fermentation conditions for enhanced BC production by Gluconacetobacter hansenii NCIM 2529 were investigated under shaking conditions. The investigation on media components and culture parameters revealed that 2 % (w/v) sucrose as carbon source, 0.5 % (w/v) potassium nitrate as nitrogen source, 0.4 % (w/v) disodium phosphate as phosphate source, 0.04 % (w/v) magnesium sulfate, and 0.8 % (w/v) calcium chloride as trace elements, pH?5.0, temperature 25 °C, and agitation speed 170 rpm with 6 days of fermentation period are optimal for maximum BC production. Production of BC using optimized media components and culture parameters was 1.66 times higher (5.0 g/l) than initial non optimized media (3.0 g/l). Fourier transform infrared spectroscopy spectrum and comparison with the available literature suggests that the produced component by G. hansenii in the present study is pure bacterial cellulose. The specific action of cellulase out of the investigated hydrolytic enzymes (cellulase, amylase, and protease) further confirmed purity of the produced BC. These findings give insight into conditions necessary for enhanced production of bacterial cellulose, which can be used for a variety of applications.     

Biological Real-Time Reaction Calorimeter Studies for the Production of Penicillin G Acylase from Bacillus badius

Penicillin G acylase (PGA) is a commercially important enzyme that cleaves penicillin G to 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PAA). The strain Bacillus badius has been identified as potential producer of PGA. A detailed calorimetric investigation on PGA production was carried out to enable generation of thermokinetic data possible for commercial application. Reaction calorimetric studies coupled with respirometric studies suggested that enzyme activity of the species B. badius was calorimetrically traceable. Three phases of growth were distinctly noticeable in the metabolic heat-time curve. Increase in enzymatic activity with restricted growth confirmed intracellular nature of the production process. The estimated heat yields due to biomass growth, 10.026 kJ/g, substrate consumption 22.761 kJ/g, and oxygen uptake 383?±?10 kJ/mol helped to understand the energetic of the organism under study. Low oxycalorific coefficient confirmed the existence of fermentation-coupled metabolism of B. badius.     

Elucidating the Effect of Glycerol Concentration and C/N Ratio on Lipid Production Using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The high demand for renewable energy and increased biodiesel production lead to the surplus availability of crude glycerol. Due to the above reason, the bio-based value addition of crude glycerol into various bioproducts is investigated; among them, microbial lipids are attractive. The present study was dedicated to find the optimal glycerol concentration and carbon/nitrogen (C/N) ratio to produce maximum lipid using Yarrowia lipolytica SKY7. The glycerol concentration (34.4 to168.2 g/L) and C/N ratio (25 to 150) were selected to investigate to maximize the lipid production. Initial glycerol concentration 112.5 g/L, C/N molar ratio of 100, and with 5 % v/v inoculum supplementation were found to be optimum for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8 % w/w with a biomass concentration of 14.8 g/L was achieved. In the case of glycerol concentration, the maximum Yp/s (0.192 g/g); Yx/s (0.43 g/g) was noted when the initial glycerol concentration was 112.5 g/L with C/N molar ratio 100 and inoculum volume 5 % v/v. The glycerol uptake was also noted to increase with the increase in glycerol concentration. At low C/N ratio, the glycerol consumption was found to be high (79.43 g/L on C/N 25) whereas the glycerol consumption was observed to decrease when the C/N ratio was raised to 150 (40.8 g/L).     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

<Emphasis Type="Italic">Hypericum japonicum:</Emphasis> a Double-Headed Sword to Combat Vector Control and Cancer

Weissella cibaria RBA12 produced a maximum of 9 mg/ml dextran (with 90% efficiency) using shake flask culture under the optimized concentration of medium components viz. 2% (w/v) of each sucrose, yeast extract, and K₂HPO₄ after incubation at optimized conditions of 20 °C and 180 rpm for 24 h. The optimized medium and conditions were used for scale-up of dextran production from Weissella cibaria RBA12 in 2.5-l working volume under batch fermentation in a bioreactor that yielded a maximum of 9.3 mg/ml dextran (with 93% efficiency) at 14 h. After 14 h, dextran produced was utilized by the bacterium till 18 h in its stationary phase under sucrose depleted conditions. Dextran utilization was further studied by fed-batch fermentation using sucrose feed. Dextran on production under fed-batch fermentation in bioreactor gave 35.8 mg/ml after 32 h. In fed-batch mode, there was no decrease in dextran concentration as observed in the batch mode. This showed that the utilization of dextran by Weissella cibaria RBA12 is initiated when there is sucrose depletion and therefore the presence of sucrose can possibly overcome the dextran hydrolysis. This is the first report of utilization of dextran, post-sucrose depletion by Weissella sp. studied in bioreactor.     

Lipase Production by Botryosphaeria ribis EC-01 on Soybean and Castorbean Meals: Optimization, Immobilization, and Application for Biodiesel Production

The effects of soybean and castorbean meals were evaluated separately, and in combinations at different ratios, as substrates for lipase production by Botryosphaeria ribis EC-01 in submerged fermentation using only distilled water. The addition of glycerol analytical grade (AG) and glycerol crude (CG) to soybean and castorbean meals separately and in combination, were also examined for lipase production. Glycerol-AG increased enzyme production, whereas glycerol-CG decreased it. A 2⁴ factorial design was developed to determine the best concentrations of soybean meal, castorbean meal, glycerol-AG, and KH₂PO₄ to optimize lipase production by B. ribis EC-01. Soybean meal and glycerol-AG had a significant effect on lipase production, whereas castorbean meal did not. A second treatment (2² factorial design central composite) was developed, and optimal lipase production (4,820 U/g of dry solids content (ds)) was obtained when B. ribis EC-01 was grown on 0.5 % (w/v) soybean meal and 5.2 % (v/v) glycerol in distilled water, which was in agreement with the predicted value (4,892 U/g ds) calculated by the model. The unitary cost of lipase production determined under the optimized conditions developed ranged from US$0.42 to 0.44 based on nutrient costs. The fungal lipase was immobilized onto Celite and showed high thermal stability and was used for transesterification of soybean oil in methanol (1:3) resulting in 36 % of fatty acyl alkyl ester content. The apparent K _m and V _max were determined and were 1.86 mM and 14.29 μmol min^?1 mg^?1, respectively.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Bioethanol Production from Soybean Residue via Separate Hydrolysis and Fermentation

Bioethanol was produced using polysaccharide from soybean residue as biomass by separate hydrolysis and fermentation (SHF). This study focused on pretreatment, enzyme saccharification, and fermentation. Pretreatment to obtain monosaccharide was carried out with 20% (w/v) soybean residue slurry and 270 mmol/L H₂SO₄ at 121 °C for 60 min. More monosaccharide was obtained from enzymatic hydrolysis with a 16 U/mL mixture of commercial enzymes C-Tec 2 and Viscozyme L at 45 °C for 48 h. Ethanol fermentation with 20% (w/v) soybean residue hydrolysate was performed using wild-type and Saccharomyces cerevisiae KCCM 1129 adapted to high concentrations of galactose, using a flask and 5-L fermenter. When the wild type of S. cerevisiae was used, an ethanol production of 20.8 g/L with an ethanol yield of 0.31 g/g consumed glucose was obtained. Ethanol productions of 33.9 and 31.6 g/L with ethanol yield of 0.49 g/g consumed glucose and 0.47 g/g consumed glucose were obtained in a flask and a 5-L fermenter, respectively, using S. cerevisiae adapted to a high concentration of galactose. Therefore, adapted S. cerevisiae to galactose could enhance the overall ethanol fermentation yields compared to the wild-type one.     

Molecular Identification Using ITS Sequences and Genome Shuffling to Improve 2-Deoxyglucose Tolerance and Xylanase Activity of Marine-Derived Fungus, Aspergillus Sp. NRCF5

During the screening of xylanolytic enzyme from marine-derived fungi isolated from the inner tissue of Egyptian soft coral Rhytisma sp., one strain, NRCF5, exhibited high enzyme activity with 0.1?% (w/v) antimetabolite 2-deoxyglucose (2DG) tolerance. This fungal strain was identified as Aspergillus sp. NRCF5 based on its morphological characteristics and internal transcribed spacer (ITS) sequences. The ITS region of hyperactive xylanolytic strain (NRCF5) was amplified, sequenced, and submitted to GenBank (accession no. JQ277356). To apply the fundamental principles of genome shuffling in breeding of xylanase-producing fungi, marine-derived fungus Aspergillus sp. NRCF5 was used as starting strain in this work and applied for induction of genetic variability using different combinations and doses of mutagens. Five mutants with high xylanase activity and 0.25?% (w/v) antimetabolite 2DG tolerance were obtained from the populations generated by the mutation of combination between ultraviolet irradiation (UV, 5?min) and N-methyl-N-nitro-N-nitrosoguanidine (NTG, 100???g/ml) for 30 (UNA) and 60 (UNB)?min as well as NTG (100???g/ml) and ethidium bromide (250???g/ml) for 30 (NEA) and 60 (NEB)?min. Then, they were subjected for recursive protoplast fusion. Seven hereditarily stable recombinants with high xylanase activity and 1.0?% (w/v) 2DG tolerance were obtained by four rounds of genome shuffling. Among them, a high xylanase-producing recombinant, R4/31, was obtained, which produced 427.5?U/ml xylanase. This value is 6.13-fold higher than that of the starting strain NRCF5 and 2.48-fold higher than that of the parent strain (mutant NEA51). The subculture experiments indicated that the high producer of marine Aspergillus sp. R4/31 fusant was stable.     

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of l-arabinose, 4-O-methyl-d-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50?°C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5–3.5% (w/v), enzyme load 40–80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2² factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.     

Acetaldehyde Detoxification Using Resting Cells of Recombinant Escherichia coli Overexpressing Acetaldehyde Dehydrogenase

Acetaldehyde dehydrogenase (E.C. 1.2.1.10) plays a key role in the acetaldehyde detoxification. The recombinant Escherichia coli cells producing acetaldehyde dehydrogenase (ist-ALDH) were applied as whole-cell biocatalysts for biodegradation of acetaldehyde. Response surface methodology (RSM) was employed to enhance the production of recombinant ist-ALDH. Under the optimum culture conditions containing 20.68 h post-induction time, 126.75 mL medium volume and 3 % (v/v) inoculum level, the maximum ist-ALDH activity reached 496.65?±?0.81 U/mL, resulting in 12.5-fold increment after optimization. Furthermore, the optimum temperature and pH for the catalytic activity of wet cells were 40 °C and pH 9.5, respectively. The biocatalytic activity was improved 80 % by permeabilizing the recombinant cells with 0.075 % (v/v) Triton X-100. When using 2 mmol/L NAD⁺ as coenzyme, the permeabilized cells could catalyze 98 % of acetaldehyde within 15 min. The results indicated that the recombinant E. coli with high productivity of ist-ALDH might be highly efficient and easy-to-make biocatalysts for acetaldehyde detoxification.     

Optimization of Levan Production by Cold-Active <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> ANT 179 and Fructooligosaccharide Synthesis by Its Levansucrase

Fructooligosaccharides (FOS) and levan attract much attention due to a wide range of applications in food technology and pharmaceutical and cosmetic industry. Bacillus licheniformis ANT 179, isolated from Antarctica soil, produced levansucrase and levan in a medium containing sucrose as carbon substrate. In this study, characterization of levansucrase and production of short-chain FOS and levan were investigated. Temperature and pH optimum of the enzyme were found to be 60 °C and pH 6.0, respectively. The optimization of fermentation conditions for levan production using sugarcane juice by response surface methodology (RSM) was carried out. Central composite rotatable design was used to study the main and the interactive effects of medium components: sugarcane juice and casein peptone concentration on levan production by the bacterium. The optimized medium with sugarcane juice at 20 % (v/v) and casein peptone at 2 % (w/v) was found to be optimal at an initial pH of 7.0 and incubation temperature of 35 °C for 48 h. Under these conditions, the maximum levan concentration was 50.25 g/L on wet weight basis and 16.35 g/L on dry weight basis. The produced inulin type FOS (kestose and neokestose) and levan were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) analysis. The study revealed that the levansucrase could form FOS from sucrose. The locally available low-cost substrate such as sugarcane juice in the form of a renewable substrate is proposed to be suitable even for scale-up production of enzyme and FOS for industrial applications. The levan and FOS synthesized by the bacterium are suitable for food applications and biomedical uses as the bacterium has GRAS status and devoid of endotoxin as compared to other Gram-negative bacteria.     

Ionic liquid-based headspace single-drop microextraction coupled to gas chromatography for the determination of chlorobenzene derivatives

A novel method, based on the coupling of ionic liquid-based headspace single-drop microextraction (SDME) with gas chromatography (GC), is developed for the determination of chlorobenzene derivatives. For the SDME of five chlorobenzene derivatives, a 1.0 μL 1-octyl-3-methylimidazolium hexafluorophosphate microdrop is exposed for 20 min to the headspace of a 15 ml aqueous sample containing 20% (w/v) NaCl placed in 25 ml vial at 40 °C. Then, the extractant is directly injected into the injector block of the GC instrument. To avoid ionic liquid leaking into the chromatographic column, a small glass tube is placed in the injection block. Under optimized operation conditions, linear relation between peak areas and analyte concentrations up to 1.5 mg L^?1 has been obtained The detection limits range from 0.1 to 0.5 μg L^?1 for the various analytes. The relative standard deviations at 1.0 μg L^?1 range from 7.7 to 12.4%, and the enrichment factors from 41 to 127. The method is simple and sensitive, and does not suffer from the influence of a solvent peak. Its applicability is demonstrated by the determination of chlorobenzenes in wastewater samples.     

Determination of Poricoic Acid A in Rat Plasma after Oral and Intravenous Administration by LC–MS–MS and Its Application to a Pharmacokinetic Study

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed and validated for the quantification of poricoic acid A (PAA) in rat plasma. The plasma samples were precipitated by protein precipitation with methanol. Glycyrrhetic acid was used as the IS. Chromatography was performed on a Dionex C₁₈ 120 Å (4.6 × 250 mm, 5 μm) column with the mobile phase composed of acetonitrile–water (90:10, v/v) at a flow rate of 0.8 mL min^?1. A tandem mass spectrometer equipped with an ESI source was used as the detector and was operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 497.4 → 423.3 and m/z 469.2 → 425.1 for PAA and IS, respectively. The calibration curves were linear over the range of 5–5,000 ng mL^?1 (r ² = 0.9966) and the lower limit of quantification (LLOQ) was 5 ng mL^?1. In this range, RSDs were <10% for intra-assay and inter-assay precisions. The accuracy expressed by deviation (DEV) was <6%, and the extraction recoveries of QC samples were >78%. The validated method was successfully used to study the pharmacokinetics of PAA in rats after intravenous administration at a dose of 1.0, 2.5 and 5.0 mg kg^?1 and oral administration at a dose of 25, 50 and 100 mg kg^?1, respectively. The relative bioavailability of PAA in rats following oral administration was achieved.     

Evaluation of a Vancomycin-Based LC Column in Enantiomeric Separation of Atenolol: Method Development, Repeatability Study and Enantiomeric Impurity Determination

An LC method was developed and validated for the enantioselective separation and enantiomeric impurity quantitation of atenolol. Separation of the atenolol enantiomers on the Chirobiotic V2 (150 mm × 4.6 mm, 5 μm) column was best achieved using a ternary mobile phase of methanol–acetonitrile-triethylamine acetate 0.5% (w/v), pH 4.5 in a ratio of (45:50:5; v/v/v). Good resolution value of R _s  = 3 was obtained at a flow rate of 1 mL min^?1 within a total run time of less than 40 min. Peak identification was achieved using the standard reference of individual enantiomers. The peak of the impurity was eluted in front of the peak of the main enantiomer. Detection was performed by UV at 226 nm. Within and between day’s repeatabilities for both retention time and peak area were investigated at three concentration levels and found to be low. The method was also found to be efficient for the determination of atenolol enantiomeric impurity. An impurity quantitation level of (R)-atenolol down to 0.08% relative to the main enantiomer (S)-atenolol was found possible.     

HPLC Method for Chiral Separation and Quantification of Antidepressant Citalopram and Its Precursor Citadiol

HPLC method enabling chiral separation and determination of citalopram (CIT), a widely used antidepressant, and its synthetic precursor citadiol in one analysis was developed and validated. Moreover, supercritical fluid chromatography was also tested and was proved to be less effective for this separation purpose. The optimized HPLC system was composed of Chiralcel OD-H column and n-hexane/propane-2-ol/triethylamine 96/4/0.1 (v/v/v) as mobile phase, column temperature 25 °C, flow rate 1.0 mL min^?1, UV detection at 250 nm. The effects of amount of propane-2-ol, triethylamine addition, and temperature on enantioselectivity and resolution of the enantiomers were evaluated. The method was found to be suitable for determination of the enantiomeric purity of CIT in bulk drugs. Enantiomers of CIT were determined in two commercially available pharmaceuticals.     

Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at ?20 or at ?80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and Na₂SO₄ (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na₂SO₄ (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na₂SO₄ are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.     

Stability-Indicating RP-HPLC Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in Dosage Forms

A new stability-indicating high-performance liquid chromatographic method has been developed for simultaneous analysis of metformin hydrochloride (MET) and sitagliptin phosphate (SIT) in pharmaceutical dosage forms. Chromatographic separation was achieved on a C₈ column. The mobile phase was methanol–water 45:55 % (v/v) containing 0.2 % (w/v) n-heptanesulfonic acid and 0.2 % (v/v) triethylamine; the pH was adjusted to 3.0 with orthophosphoric acid. The flow rate was 1 mL min^?1 and the photodiode-array detection wavelength was 267 nm. The linear regression coefficients for metformin and sitagliptin were 0.9998 and 0.9996 in the concentration ranges 50–450, and 10–150 μg mL^?1, respectively. The relative standard deviations for intra and inter-day precision were below 1.5 %. The drugs were subjected to a variety of stress conditions—acidic and basic hydrolysis, and oxidative, photolytic, neutral, and thermal degradation. The products obtained from photolytic degradation were similar to those from neutral hydrolytic degradation and different from produced by acidic and basic hydrolysis. The method resulted in detection of 15 degradation products (D1–D15); among these, the structures of D1, D3, D9, and D13 were identified. The respective mass balance for MET and SIT was found to be close to 97.60 and 99.12 %. The specificity of the method is suitable for a stability-indicating assay.     

Improving the Determination of Nitrovin in Feeds by Reversed-Phase LC with SPE

A simple reversed-phase liquid chromatographic method with ultraviolet detector (378 nm) for the determination of nitrovin in feeds was improved and validated. The mobile phase was a mixture of acetonitrile and 0.1% formic acid solution (v/v) in the ratio of 50:50 (v/v), and the flow rate was set at 1.2 mL min^?1. The extraction solution was a mixture of dimethyl formamide, acetonitrile and methanol (50:25:25, v/v), the sample was cleaned-up with reversed-phase solid phase extraction cartridge. The standard nitrovin was purified with crude nitrovin product by ethylene glycol monoethyl ether and identified by elemental analyzer. The limit of detection was 0.05 mg kg^?1 and the limit of quatification was 0.2 mg kg^?1 in feeds. The assay had satisfactory selectivity, recovery, linearity and precise repeatability and trueness.