Deep-freeze Conditions Research Articles

Status of Infection during a Natural Outbreak of Brucellosis in Unvaccinated Buffaloes at a Livestock Farm of Madhya Pradesh in India by using Competitive Enzyme-linked Immunosorbent Assay

Background: Brucellosis is one of India’s most important bacterial diseases of large ruminants. It is of economic importance. It is caused by members of the Brucella species and responsible for abortion in pregnant bovines usually during the first term and generally after the fifth month of gestation. Hence, livestock farm with a history of abortion in pregnant animals has to be investigated for Brucellosis. Methods: Whole blood samples (of 50 individual buffaloes) were collected aseptically from a herd of buffaloes experiencing a natural outbreak of Brucellosis. Extracted serum samples were tested after clarification in commercially procured competitive enzyme-linked immunosorbent assay for the serological laboratory diagnosis of Brucellosis. Result: Out of 50 buffalo serum samples tested by using commercially procured competitive enzyme-linked immunosorbent assay, a total of 24 buffalo serum samples tested positive for the presence of Antibodies against Brucella species. Overall seropositivity of 48% was recorded for Brucellosis in the investigated buffalo herd experiencing a natural infection in the present study even after long-term storage of serum samples at deep freezing conditions.

Assessment of Faecal Microbiota Transplant Stability in Deep-Freeze Conditions: A 12-Month ExVivo Viability Analysis.

Faecal microbiota transplantation (FMT) is an established treatment for Clostridioides difficile infection and is under investigation for other conditions. The availability of suitable donors and the logistics of fresh stool preparation present challenges, making frozen, biobanked stools an attractive alternative. This study aimed to evaluate the long-term viability of bacterial populations in faecal samples stored at -80°C for up to 12 months, supporting the feasibility of using frozen grafts for FMT. Fifteen faecal samples from nine healthy donors were processed, mixed with cryoprotectants and stored at -80°C. Samples were assessed at baseline and after 3, 6 and 12 months using quantitative culturing methods to determine the concentration of live bacteria. Quantitative analysis showed no significant decrease in bacterial viability over the 12-month period for both aerobic and anaerobic cultures (p = 0.09). At all timepoints, the coefficients of variability in colony-forming unit (CFU) counts were greater between samples (102 ± 21% and 100 ± 13% for aerobic and anaerobic cultures, respectively) than the variability between measurements of the same sample (30 ± 22% and 30 ± 19%). The study confirmed that faecal microbiota can be preserved with high viability in deep-freeze storage for up to a year, making allogenic FMT from biobanked samples a viable and safer option for patients. However, a multidonor approach may be beneficial to mitigate the risk of viability loss in any single donor sample.

Stability of pesticide residues in citrus fruits during storage

Introduction. Storage of selected samples before transportation to the laboratory and immediately before analysis is an important component in the process of product quality control and evaluation.
 Purpose of the work was to evaluate the degradation of pesticide active substances in real samples of citrus fruit left for long periods of storage in deep freezing conditions.
 Materials and methods. High-performance liquid chromatography with triple quadrupole mass detector (HPLC-MS/MS) was used to identify and quantify pesticide active substances. According to the method of analysis MUK 4.1.3657–20, cryogenic grinding of samples was carried out in a cutter using dry ice. Sample preparation was carried out using the QuEChERS technology. To extract analytes from a homogenized sample acetonitrile was used in the presence of salts containing citrate buffer, followed by purification of the extract by SPE.
 Results. In the studied samples of citrus, the analytes showed stability. Thirty months after storage of samples in deep freezing conditions (temperature not higher than –20 °C), the identified levels of active ingredients of the pesticides imazalil, pyrimethanil and prochloraz did not change by more than 20% compared to the previously detected concentrations. Insignificant amounts of imidacloprid, thiabendazole and pyriproxyfen, as well as traces of azoxystrobin and trifloxystrobin, detected in the study of samples, were also found in samples after long-term storage. 
 Limitation. The study is limited to the study of the stability of individual active substances found in real samples of citrus fruits intended for sale to the consumer.
 Conclusion. Proper storage of samples should ensure the preservation of the levels of active substances in the samples and exclude their change due to various processes (volatilization, enzymatic and hydrolytic decomposition, etc.), which requires the performance of preliminary experimental studies to assess their stability. The work showed the stability of residual quantities of imazalil, pyrimethanil, prochloraz, imidacloprid, thiabendazole, and pyriproxyfen in citrus fruit matrices under storage conditions in a freezer at not more than –20 °C for 30 months.

A Sensitive Liquid Chromatography-Mass Spectrometry Method for Determination of Toosendanin in Rat Plasma and its Application to Pharmacokinetic Study.

A simple, rapid and sensitive analytical method was developed for the determination of toosendanin in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Andrographolide was selected as the internal standard, and the plasma samples were extracted by liquid-liquid extraction with diethyl ether. Chromatographic separation was performed on a Dikma Spursil C18, 3.5μm (150 × 2.1mm i.d) analytical column with 85% methanol:water (v/v) containing 0.025% formic acid (pH = 3.9) as mobile phase. The flow rate was 0.25mL/min, and the total run time was 3min. Detection was performed with a triple-quadrupole tandem mass spectrometer using negative ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 573.1→531.1 and 349.0→287.0 for toosendanin and andrographolide, respectively. Good linearity was observed over the concentration range of 3.125-500ng/mL in 100μL of rat plasma with a correlation coefficient ˃0.9997. Intra- and inter-assay variabilities were ˂8.5% in plasma. The recovery and the matrix effect were in the range 71.8-73.5% and 96.4-103.8%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw settings, in the autosampler, and under deep-freeze conditions). The method was successfully applied to a pharmacokinetic study of toosendanin after its oral administration in rats at a dose of 10mg/kg.

Влияние замораживания семян кенафа (Hibiscus cannabinus L.) на всхожесть, рост и развитие растений

Abstract A comparative study of the effect of different storage regimes of seeds (at + 5 ° С, -20 ° С and under deep freezing conditions at -196 ° С) of a valuable bast fiber culture - kenaf - Hibiscus cannabinus L. on their viability and the development of plants from them in field conditions. Freezing increases the laboratory and field germination of kenaf seeds, and also increases the energy of their germination. It did not cause the appearance of non-viable, ugly seedlings and plants, as well as their death. Plants went through a full development cycle and set full-fledged seeds. Biometric parameters of plants obtained from thawed seeds and control ones are very close. Cryopreservation is a promising way of storing seeds of the studied species, since it ensures longer periods of preservation of their viability. Keywords: KENAF, SEED BANKS, LONG-TERM SEED STORAGE REGIMES, CRYOCONSERVATION

Влияние замораживания семян Abutilon theophrasti Medik. и Eruca vesicaria (L.) Cav. на всхожесть, рост и развитие растений

A comparative study of the effect of different storage regimes of seeds (at + 5 ° С, -20 ° С and under deep freezing conditions at -196 ° С) of two cultivated plants of the Abutilon theophrasti Medik. and Eruca vesicaria (L.) Cav. on their viability and the development of plants from them in the field was carried out. Freezing increases the laboratory and field germination of the seeds of the A. theophrasti and does not significantly affect the germination of the seeds of the A. theophrasti, and also increases the germination energy of the seeds of both plant species. It did not cause the appearance of non-viable, ugly seedlings and plants. Plants went through a full development cycle and set full-value seeds. Biometric parameters of plants obtained from thawed seeds and control ones are very close. Cryopreservation is a promising way of storing seeds of the studied species, since it ensures longer periods of preservation of their viability. Keywords: ABUTILON THEOPHRASTI; ERUCA VESICARIA; SEED BANKS; LONG-TERM SEED STORAGE REGIMES; CRYOPRESERVATION

Scouring by rafted ice and cryogenic patterned ground preserved in a Palaeoproterozoic equatorial proglacial lagoon succession, eastern India, Nuna supercontinent

The Chaibasa Formation contains the oldest known record of ploughmarks formed by rafted ice preserved on Earth. The Palaeoproterozoic (Orosirian-Rhyacian, 1.86–2.1 Ga) Chaibasa Formation in the Dhalbhumgarh area, eastern India, represents sedimentation in an ice-contact proglacial lagoon affected by iceberg (bits and growlers) calving and lake-ice rafting. Ice scouring, both on muddy and sandy substrates, as well as ice “rosettes” developed as consequence of the ploughing action of floating icebergs that were moved by wind, meltwater floods and tidal currents on soft sediments. Muddy flats surrounding the proglacial lagoon underwent seasonal deep freezing conditions developing cryogenic patterned ground containing reticulate ice, ice veins and sand wedges, suggesting the occurrence of proglacial permafrost areas coeval with Proterozoic glaciers. The central part of the proglacial lagoon was characterized by deposition of glacial varves punctuated by pebble and cobble size angular dropstones accumulated in the basin as ice-rafted debris from floating ice. Evidences provided in this paper reinforced the idea that 1.86–2.1 Ga ago glacial conditions prevailed after the supposed ending of the Huronian glaciation. This paper provides detailed examples that can be used to recognize possible floating/drifted ice scours in other Precambrian successions, as these elements have largely passed undetected, being probably widely reported in the literature as generic soft-sediment deformation structures. Similar soft-sediment deformations from Precambrian clastic depositional systems should be re-evaluated as there is a significant possibility that most of the Precambrian iceberg-related structures had escape notice, as originally suggested Eyles et al. in 1997.

Development and validation of novel LC–MS/MS method for determination of Lusutrombopag in rat plasma and its application to pharmacokinetic studies

A novel, simple and sensitive method for the determination of Lusutrombopag in rat plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated. The determination was performed on an API4000 triple quadrupole mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]+ ions m/z 593.1 → 272.3 for Lusutrombopag. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 2.0 ng/mL in rat plasma. Good linearity was obtained over the range of 2.0–150.0 ng/mL and the correlation coefficient was found to be 0.9998. The intra and inter-day precisions were found to be 3.8–6.9% and 6.8–10.5%, respectively. The intra and inter-day accuracy derived from QC samples was found to be 2.5–4.9% and 5.5–7.2%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw, in the autosampler and under deep-freeze conditions). The F-test and t-test at 95% confidence level were subjected on data for statistical analysis. The developed method was successfully applied to the pharmacokinetic study in rats.

Response of Methanogenic Archaea from Siberian Permafrost and Non-permafrost Environments to Simulated Mars-like Desiccation and the Presence of Perchlorate.

Numerous preflight investigations were necessary prior to the exposure experiment BIOMEX on the International Space Station to test the basic potential of selected microorganisms to resist or even to be active under Mars-like conditions. In this study, methanogenic archaea, which are anaerobic chemolithotrophic microorganisms whose lifestyle would allow metabolism under the conditions on early and recent Mars, were analyzed. Some strains from Siberian permafrost environments have shown a particular resistance. In this investigation, we analyzed the response of three permafrost strains (Methanosarcina soligelidi SMA-21, Candidatus Methanosarcina SMA-17, Candidatus Methanobacterium SMA-27) and two related strains from non-permafrost environments (Methanosarcina mazei, Methanosarcina barkeri) to desiccation conditions (-80°C for 315 days, martian regolith analog simulants S-MRS and P-MRS, a 128-day period of simulated Mars-like atmosphere). Exposure of the different methanogenic strains to increasing concentrations of magnesium perchlorate allowed for the study of their metabolic shutdown in a Mars-relevant perchlorate environment. Survival and metabolic recovery were analyzed by quantitative PCR, gas chromatography, and a new DNA-extraction method from viable cells embedded in S-MRS and P-MRS. All strains survived the two Mars-like desiccating scenarios and recovered to different extents. The permafrost strain SMA-27 showed an increased methanogenic activity by at least 10-fold after deep-freezing conditions. The methanogenic rates of all strains did not decrease significantly after 128 days S-MRS exposure, except for SMA-27, which decreased 10-fold. The activity of strains SMA-17 and SMA-27 decreased after 16 and 60 days P-MRS exposure. Non-permafrost strains showed constant survival and methane production when exposed to both desiccating scenarios. All strains showed unaltered methane production when exposed to the perchlorate concentration reported at the Phoenix landing site (2.4 mM) or even higher concentrations. We conclude that methanogens from (non-)permafrost environments are suitable candidates for potential life in the martian subsurface and therefore are worthy of study after space exposure experiments that approach Mars-like surface conditions.

Electrochemical behaviors of Li-argyrodite-based all-solid-state batteries under deep-freezing conditions.

We probe the electrochemical performance of Li-argyrodite-based all-solid-state batteries under deep-freezing conditions (-30 °C) using electrochemical impedance spectroscopy. The performance deterioration is mainly caused by the increased interfacial resistances of electrolyte and active materials resulting from the slow kinetics of Li-ion transport in solid materials at low temperatures.

Quantification of Tamoxifen Polymeric Nanoparticles in female rodent breast tissue by UPLC/ESI-Q-TOF MS/MS

Objective: The present work aims to develop and validate stability indicating a novel liquid chromatography method with applicability i.e. to study the pharmacokinetics factor as well as estimate Tamoxifen (TMX) in bio-analytes, using Ultra High Pressure Liquid Chromatography/ Mass Spectrometry (UPLC-ESI-Q-TOF-MS/MS) in plasma. Methods: A bioanalytical method based on UPLC-ESI-Q-TOF-MS/MS has been developed and validated for the quantitative determination of TMX in female mice plasma using Clomiphine as an Internal Standard (IS). After Liquid-liquid extraction (LLE), analyte and IS, chromatographic separation was achieved on ACQUITY UPLC BEH C18 Waters column with dimensions; 100 mm × 2.1 mm; 1.7 μm, isocratic mobile phase (Acetonitrile:2 mM ammonium formate: 90:10; v/v), and a flow rate of 0.25 mL min -1 . Results: The transitions occurred at m/z 372.1→178.1 and m/z 407.1 → 100.1 for TMX and IS, respectively. Liquidliquid extraction technique (LLE) using ethyl acetate was applied in order to optimize the recovery of analytes in mice plasma. The run and retention time of TMX were 6.0 and 2.63 minutes, respectively while the linear dynamic range established was 1.002-4001.07 ng/ml (r2>0.998 ± 0.0003). Intra-assay and inter-assay accuracy (% RSD) was found in the range; 2.43- 3.49. Conclusion: The proposed LC–MS/MS assay method is simple, rapid and sensitive for the determination of TMX in mice plasma for pharmacokinetic studies. Analytes were stable under various conditions i.e. autosampler, freeze–thaw, at room temperature, and under deep freeze conditions. Key words Tamoxifen, Breast Cancer, UPLC/ESI-Q-TOF-MS/MS method Validation, Polymeric nanoparticles, Pharmacokinetics.

The German Environmental Specimen Bank

The main objective of the German Environmental Specimen Bank (ESB) is the long-term storage of environmental and human samples under stable deep-freeze conditions for future research. The ESB is unique in providing a continuous historical record of environmental and human exposure to chemicals in Germany. ESB was started parallel to the development of the first German Chemicals Legislation in the late 1970s. In 1979, the ESB test operation began. After the Chemicals Law came into force in 1982, the ESB was established as a permanent facility in 1985. With the new European Chemicals Legislation, REACH, in 2007 responsibility for the safety of commercial chemicals and risk assessment was assigned to the industry. Since then, the ESB has become even more important in verifying the self-assessment of the industry, in evaluating the effectiveness of regulations, thus ensuring the protection of humans and the environment against adverse effects caused by exposure to chemicals. These objectives are pursued by the regular monitoring of contaminations and the assessment of temporal trends. Demonstrating the necessity of deriving exposure reduction measures, ESB results serve as key information for policy-makers. Information on preventing exposure to chemicals is available to the general public and to the public health services. The ESB is thus an important monitoring instrument of the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety. The Federal Environment Agency operates the ESB based on its own concepts, heads the scientific data evaluation and transfers results into the environmental policy arena and to the general public.

English

A field experiment was carried out to assess the effects of vesicular arbuscular mycorrhiza (VAM), Organo-mineral fertilizer (OMF) and NPK (15:15:15), on the performance and post-harvest quality of sweet corn. The experiment was laid out in a split plot design with mycorrhiza as main plot and fertilizer combinations as treatments. VAM inocula were applied at a rate of 888.88 kg ha-1, OMF at 2000 kg ha-1 and NPK (15:15:15) at 400kg ha-1. Storability of the sweet corn was evaluated at open shelf (25-27°C), normal refrigeration (40°C) and deep freezing conditions (0°C). Results showed that there were significant differences (p <0.05) between mycorrhiza and non mycorrhiza. Across mycorrhiza treatment, the highest yields of free corn ears were obtained with the complementary use of OMF and NPK (15:15:15). Fertilizer application had significant effect on the storage life of sweet corn, however, sweet corn stored by freezing at 0°C had the best storage properties in terms of firmness, weight loss and disease incidence.   Key words: Vesicular arbuscular mycorrhiza; sweetcorn yield; Organo-mineral fertilizer.

Jumplike Deformation as the Scale Effect Measure for the Metal Deformed Volume Under Deep-Freezing Conditions: Experiment and Modeling

Tensile tests of titanium alloy and austenitic steel specimens have been conducted in the range of temperatures 300–4 K. Under deep-freezing conditions, there occurs a new type of scale effect – variation of low-temperature jumplike deformation, which causes a sharp variation of the plasticity characteristic (the relative elongation). The difference between jumplike deformation values in the maximum and minimum cross-sectional areas of standard cylindrical specimens is introduced as the scale effect measure. Via mathematical modeling performed for 03Kh20N16AG6 steel, we have obtained dependencies of the scale effect on the most critical factors – the trigger stress of deformation jump and specimen–test machine system stiffness. The scale effect nature is studied, and options of its minimization, in reference to the standardization of mechanical tests, are discussed.

DOE-Based Stability Indicating RP-HPLC Method for Determination of Lacidipine in Niosomal Gel in Rat: Pharmacokinetic Determination

Lacidipine (LAC) is a calcium channel blocker used in treatment of hypertension. In this study high performance liquid chromatography method (HPLC) is applied to the determination of LAC in rat plasma. The design of experiment used 3- factor 3- level Box Behnken Design for optimization of mobile phase condition. The effluent was monitored by UV detector at 240 nm, at a flow rate of 1.0 mL/min using amlodipine as internal standard. A linear calibration curve very well fits our data from 20-1200 ng/mL with limit of detection (LOD) quantitation (LOQ) 1.490 and 4.848 ng mL-1 respectively. The method was found to be linear, precise and accurate. Analytes were stable under various conditions (during freeze-thaw, at room temperature, and under deep freeze conditions). The pharmacokinetic study was performed after application of LAC niosomal transgel in rats and results showed enhance bioavailability by 2.57 fold as compared to oral formulation.

Development of a validated UPLC-qTOF-MS Method for the determination of curcuminoids and their pharmacokinetic study in mice

BackgroundA specific and sensitive UPLC-qTOF-MS/MS method has been developed for the simultaneous determination of curcuminoids. These Curcuminoids comprises of curcumin, a principal curcuminoid and other two namely, demethoxycurcumin, and bisdemethoxycurcumin obtained from rhizomes of Curcuma longa an ancient Indian curry spice turmeric, family (Zingiberaceae).MethodsThese analytes were separated on a reverse phase C18 column by using a mobile phase of acetonitrile: 5% acetonitrile in water with 0.07% acetic acid (75:25 v/v), flow rate of 100 μL/min was maintained. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions in the positive mode for curcuminoids were at m/z 369.1066, 339.1023 and 309.0214 respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique.ResultsThe total run time was 5 min and the peaks of the compounds, bisdemethoxycurcumin, demethoxycurcumin and curcumin occurred at 2.06, 2.23 and 2.40 min respectively. The calibration curves of bisdemethoxycurcumin, demethoxycurcumin and curcumin were linear over the concentration range of 2–1000 ng/mL (r2, 0.9951), 2–1000 ng/mL (r2, 0.9970) and 2-1000 ng/mL (r2, 0.9906) respectively.Intra-assay and inter-assay accuracy in terms of % bias for curcumin was in between −7.95to +6.21, and −7.03 to + 6.34; for demethoxycurcumin was −6.72 to +6.34, and −7.86 to +6.74 and for bisdesmetoxycurcumin was −8.23 to +6.37 and −8.47 to +7.81. The lower limit of quantitation for curcumin, demethoxycurcumin and bisdemethoxycurcumin was 2.0 ng/mL. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). This validated method was used during pharmacokinetic studies of curcumin in the mouse plasma.ConclusionsA specific, accurate and precise UPLC-qTOF-MS/MS method for the determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin both individually and simultaneously was optimized.

UHPLC/ESI‐Q‐TOF‐MS method for the measurement of dopamine in rodent striatal tissue: A comparative effects of intranasal administration of ropinirole solution over nanoemulsion

An ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method (UHPLC/ESI-Q-TOF-MS) for the analysis of dopamine (DA) in Wistar rat brain homogenate has been developed and validated. The chromatographic separation was achieved on a Waters ACQUITY UPLC™ BEH C18 (100.0 mm × 2.1 mm; 1.7 µm) column using isocratic mobile phase, consisting of acetonitrile and Formic acid (0.1% w/v) (10: 90; v/v), at a flow rate of 0.15 ml min(-1) . The transitions occurred at m/z 154.04 → 137.006 for DA, and m/z 184.204 → 166.08 for the internal standard. The recovery of the analytes from Wistar rat brain homogenate was optimized using liquid-liquid extraction technique (LLE) in ethyl acetate. The total run time was 3.5 min and the elution of DA occurred at 1.44 ± 0.05 min. The linear dynamic range was established over the concentration range 75-750 ng mL(-1) (r(2) ; 0.9921 ± 0.0005) for DA. The intra-assay and inter-assay accuracy in terms of % CV was in the range 0.73-2.80. The lower limit of detection (LOD) and quantitation (LOQ) for DA was 0.278 and 0.844 ng mL(-1) , respectively. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). The developed method was successfully applied for in vivo profiling in rodents.

Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Improved techniques to enhance the yield of paddy straw mushroom (Volvariella volvacea) for commercial cultivation

Cultivation of Volvariella volvacea in circular compact bed method recorded maximum yield of 972.8 g/bed (biological efficiency of 23.8%). Seeding the beds with 15 and 20 days old spawn with well developed chlamydospores contributed for significantly higher yields. Oil palm bunch waste in combination with rice straw or alone recorded significantly higher yields of 876.3 and 832 g/bed, and cotton waste, in combination with rice straw or alone, recorded 841.6 g and 778 g/bed, respectively. In vitro evaluation of the micronutrient based boosters VVB1 (CaCO 3 400 ppm + CaCl 2 50 ppm + KH 2 PO 4 50 ppm + NaCl 50 ppm + Na 2 HPO 4 50 ppm) and VVB2 (CaCl 2 400 ppm + Na 2 HPO 4 100 ppm) produced profuse mycelium with aerial silky strands, early chlamydospore formation (20.8 and 21.8 days) and increased chlamydospore density (7.8 × 10 2 and 7.2 × 10 2 spores/ml). Yields of 960 g/bed with more number of sturdy fruiting bodies were observed in beds sprayed with VVB1. V. volvacea can be successfully cultivated from March to September with 18 to 20% biological efficiency. Storage of V. volvacea at egg stage in air tight plastic box at deep freeze (-20°C) conditions remained intact until 36 h without spoilage. Keywords: Paddy straw mushroom, Volvariella volvacea , boosters, bed methods, cultivation techniques

An LC–MS/MS method for determination of jujuboside A in rat plasma and its application to pharmacokinetic studies

A novel, simple, and sensitive method for the determination of jujuboside A in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. Following solid-phase extraction, measurement of jujuboside A was performed by negative ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The limit of detection was 1.25ng/mL, and the lower limit of quantification was 5ng/mL in rat plasma. Good linearity was obtained over the range of 6.25–500ng/mL, and the correlation coefficient was better than 0.998. The intra- and inter-day precisions ranged 4.4–7.5% and 2.9–10.7%, respectively. The accuracy derived from QC samples ranged 3.2–7.8% and 2.2–3.5%, respectively. The recovery ranged from 72.9 to 75.1% and the matrix effect from 96.7 to 105.3%. The analyte was stable under various conditions (at room temperature, during freeze–thaw, in the autosampler and under deep-freeze conditions). The developed method was successfully applied to the pharmacokinetic study in rats.