Diester Research Articles

Flow characteristics of integrated motor-pump assembly with phosphate ester medium for aerospace electro-hydrostatic actuators

Motor-pump assembly is the core component of the Aerospace Electro-hydrostatic Actuator (EHA). Thus, the design of the motor pump can be very challenging under conditions of high speed and wide pressure range, especially in particular working mediums. Our ultimate goal is to pursue better flow characteristics under a wide range of working conditions. In this paper, we built a sub-model of the main friction interfaces and a model of single-shaft coaxial motor-pump assembly adopting the method of hierarchical modeling. The experimental investigation of the output characteristics was mainly carried out in a phosphate ester medium environment. Then, the flow characteristics were compared and analyzed with the simulation results. Results indicated that the flow characteristics of the motor-pump assembly could be accurately simulated by the model and quite severe in a low speed and high-pressure environment.

Cotton-maize intercropping increases rhizosphere soil phosphorus bioavailability by regulating key phosphorus cycling genes in northwest China

Intercropping is widely used as an important means to promote crop productivity; but the underpinning mechanisms of rhizosphere soil microbial processes in shaping soil phosphorus (P) bioavailability under cotton-based intercropping systems have not been investigated in arid northwest China. Therefore, taking cotton-maize intercropping as an example, biologically based P (BBP) and metagenomics methods were used to investigate changes of soil P bioavailability, microbial community and functional P-cycling genes in the rhizosphere. Three treatments were cotton monoculture, maize monoculture and cotton-maize intercropping. We found that rhizosphere soils from cotton-maize intercropping exhibited higher soil organic matter (SOM), available P (AP), and alkaline phosphatase (ALP) than those of crop monocultures (p < 0.05). The change of bioavailable-P fractions in the order HCl-P > Citrate-P > Enzyme-P > CaCl2-P, and higher mount of CaCl2-P, Citrate-P and Enzyme-P were observed in soil samples from cotton-maize intercropping. These results indicated that P bioavailability were improved after two crops intercropping. The dominate soil microbial community from all treatments belonged to the phyla Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi and Gemmatimonadetes. In addition, we observed that the expression of functional genes which involved in soil microbial inorganic phosphate transportation (phoR), inorganic phosphate solubilization (phoD, glpA, glpK, glpQ), phosphate ester mineralization (gcd, ppk, ppx), and phosphate ester mineralization (pstABC, pit) were also up-regulated in cotton-maize intercropping (p < 0.05). Redundancy analysis showed that SOM, CaCl2-P and AP significantly correlated genes gcd, ppx, pstABC, pit, and phoD, suggesting that SOM, CaCl2-P and available P are important factors in influencing expression of these P-cycling functional genes. It is concluded that cotton-maize intercropping increased P bioavailability via changing rhizosphere soil microbial composition and functional genes.

Room Temperature DBN Initiated Phospha‐Brook Rearrangement of α‐Hydroxyphosphonates to Phosphates

AbstractA series of substituted aryl phosphate esters have been synthesized from their α‐hydroxyphosphonates substrates, using DBN (1,5 diazabicyclo(4.3.0)non‐5‐ene) at room temperature, via a phospha‐Brook rearrangement. The aryl‐substrate dependence of the rearrangement was explored, and excellent yields of the phosphate esters were achieved irrespective of whether the aryl moiety was activated or unactivated. A plausible mechanism for the rearrangement has been proposed. Based on the low temperature 31P‐NMR, the mechanism of the phospha‐Brook rearrangement is proposed to take place via an oxaphosphirane intermediate.

Review of preclinical data of PF-07304814 and its active metabolite derivatives against SARS-CoV-2 infection

Main protease (Mpro) is a superior target for anti-SARS-COV-2 drugs. PF-07304814 is a phosphate ester prodrug of PF-00835231 that is rapidly metabolized into the active metabolite PF-00835231 by alkaline phosphatase (ALP) and then suppresses SARS-CoV-2 replication by inhibiting Mpro. PF-07304814 increased the bioavailability of PF-00835231 by enhancing plasma protein binding (PPB). P-glycoprotein (P-gp) inhibitors and cytochrome P450 3A (CYP3A) inhibitors increased the efficacy of PF-00835231 by suppressing its efflux from target cells and metabolism, respectively. The life cycle of SARS-CoV-2 is approximately 4h. The mechanisms and efficacy outcomes of PF-00835231 occur simultaneously. PF-00835231 can inhibit not only cell infection (such as Vero E6, 293T, Huh-7.5, HeLa+angiotensin-converting enzyme 2 (ACE2), A549+ACE2, and MRC-5) but also the human respiratory epithelial organ model and animal model infection. PF-07304814 exhibits a short terminal elimination half-life and is cleared primarily through renal elimination. There were no significant adverse effects of PF-07304814 administration in rats. Therefore, PF-07304814 exhibits good tolerability, pharmacology, pharmacodynamics, pharmacokinetics, and safety in preclinical trials. However, the Phase 1 data of PF-07304814 were not released. The Phase 2/3 trial of PF-07304814 was also suspended. Interestingly, the antiviral activities of PF-00835231 derivatives (compounds 5-22) are higher than, similar to, or slightly weaker than those of PF-00835231. In particular, compound 22 exhibited the highest potency and had good safety and stability. However, the low solubility of compound 22 limits its clinical application. Prodrugs, nanotechnology and salt form drugs may solve this problem. In this review, we focus on the preclinical data of PF-07304814 and its active metabolite derivatives to hopefully provide knowledge for researchers to study SARS-CoV-2 infection.

A novel polymer reactive flame retardant for the preparation of highly durable cotton fabrics

A novel polymer ammonium salt of polyethyleneimine phosphate phosphonic acid (APEMPPA) flame retardant for cotton fabrics was synthesized and characterized by nuclear magnetic resonance (NMR), in which some ammonium phosphoric acid groups were replaced by phosphate ester groups to decrease the phenomena that the ammonium ions in flame retardant would exchange with metal ions such as Ca2+ and Mg2+ in washing water to keep flame retardance well after washing. Energy dispersive X-ray (EDX), Fourier transform infrared spectroscopy (FTIR) results, and durability of treated cotton fabrics suggested that APEMPPA was grafted onto the cellulose. Limiting oxygen index (LOI) of 40 wt% APEMPPA-treated cotton fabric was 44.5 %, which was still 34.7 % after 50 LCs according to AATCC 61-2013 3A standard. Cone calorimetric and thermogravimetric (TG) results showed the treated cotton fabrics had excellent flame retardancy. TG-FTIR and Raman test results proposed that APEMPPA mainly played a condensed phase flame retardant. EDX results indicated that replacing some ammonium phosphate groups with phosphate ester groups was effective in maintaining flame retardancy during washing. All results showed that increasing the molecular weight and introducing phosphonate group in ammonium phosphorus acid flame retardant can effectively improve flame retardancy and durability. APEMPPA-treated cotton fabrics maintained good tensile strength.

Phytate-based transparent and waterproof intumescent flame-retardant coating for protection of wood

Phytic acid-based phosphate esters (PAPEGs) were prepared through the reaction between phytic acid (PA) and different proportions of polyethylene glycol 200 (PEG-200). Intumescent flame-retardant coating (IFRC) with PAPEG3, PAPEG6, PAPEG9 and PAPEG12 were obtained via thermal curing of PAPEGs and melamine formaldehyde resin (MFR), respectively. The backside temperature of steel with MFR/PAPEGs coating was much lower than that of MFR/PA coating. MFR/PAPEG6 coating showed the best flame retardancy, and the char thickness reached 48 mm from initial thickness of 0.4 mm, and the expansion ratio was 120 times. Cone calorimeter confirmed that the incorporation of PAPEG6 could minimize the heat release rates and total heat release of materials during combustion. Moreover, the MFR/PAPEGs coating possessed both excellent transparency and water resistance. The transmittance of MFR/PAPEG6 and MFR/PAPEG9 was above 87% at 600 nm, and transmittance of MFR/PAPEG12 even reached 90%. The proposed flame-retardant mechanism was that MFR/PAPEGs produced phosphorus-containing acids to dehydrate and carbonize PEG, leading to the formation of graphitized chars. Meanwhile, the nitrogen-containing intermediates simultaneously released noncombustible gases to expand the char and interrupt the combustion reaction.

Safety and efficacy of a feed additive consisting of riboflavin-5'-phosphate ester monosodium salt (vitamin B2) (from riboflavin 98%, produced by Bacillus subtilisKCCM 10445) for all animal species (Hubei Guangji Pharmaceutical Co. Ltd).

Following a request from the European Commission, the Panelon Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of vitamin B2 in the form of riboflavin-5'-phosphate ester monosodium salt. The additive is manufactured by chemical synthesis from riboflavin 98%, produced by fermentation with a genetically modified strain of Bacillus subtilis (KCCM 10445). Riboflavin 98% has been previously evaluated by the FEEDAP Panelfor its use as feed additive for all animal species. Since the additive under assessment contains the phosphate ester monosodium salt of a riboflavin (98%) preparation already considered safe, the FEEDAP Panelconcluded that the addition of the salt does not add any toxicological property to the final additive. Therefore, the additive is safe for the target species, the consumer and the environment. The additive is not a skin/eye irritant and it is not considered a respiratory sensitiser. Riboflavin is a known photosensitiser which may elicit skin and eye photoallergic reactions. The additive under assessment is effective in covering the animals' requirements for vitamin B2 when administered via feed and/or water for drinking.

Differentially expressed genes identification and bioinformatics analysis of venous blood in patients with mild preeclampsia

Background: Preeclampsia (PE) is a syndrome characterized by hypertension (systolic blood pressure ≥140 mmHg, or diastolic blood pressure ≥90 mmHg) and proteinuria that develops after 20 weeks of gestation. It is classified as mild PE and severe PE. Placental abruption, fetal growth restriction, and fetal death are common complications of PE, which is a serious threat to maternal and infant safety during pregnancy. Bioinformatics analysis can dig into the undiscovered biological information, and have a deeper understanding of the pathogenesis of diseases. At present, the pathogenesis of PE has not been fully studied. There is no information in literature on key gene screening and bioinformatics studies of mild PE. Aims and Objectives: The aim of the study was to explore the differential genes screening and related biological process (BP) in venous blood of patients with mild PE. Materials and Methods: GSE48424 dataset was downloaded from Gene Expression Omnibus database, differential genes were screened. Gene ontology (GO)|Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analysis was completed. The protein-protein interaction (PPI) network of differential genes was mapped using STRING and Cytoscape. Identify key modules and genes involved in mild PE. Results: A total of 433 up-regulated and 1242 down-regulated genes were obtained. GO function analysis showed that BP was mainly related to endosome organization, dephosphorylation, and endomembrane system organization. Cellular component is mainly related to promyelocytic leukemia body, nuclear membrane, and nuclear envelope. Molecular function is mainly related to ubiquitin-like protein transferase activity，phosphoric ester hydrolase activity, and phosphatase activity. KEGG showed that differential genes were enriched in sphingolipid, TNF, herpes simplex virus 1 infection, and pancreatic cancer pathway. The PPI network of differential genes was constructed to obtain 10 key genes involved in mild PE: SGMS1, SGPP1, ASAH1, PPAP2C, PPAP2B, PPP1R12A, WDR82, PPP2R2A, PPP4R2, and PPP4R1. Conclusion: Using bioinformatics technology to identify the hub genes involved in mild PE can provide a favorable basis for further study of biological markers and pathogenesis of mild PE.

Cooperative Catalysis between Dual Copper Centers in a Metal-Organic Framework for Efficient Detoxification of Chemical Warfare Agent Simulants.

Chemical warfare agents (CWAs) are among the most lethal chemicals known to humans. Thus, developing multifunctional catalysts for highly efficient detoxification of various CWAs is of great importance. In this work, we developed a robust copper tetrazolate metal-organic framework (MOF) catalyst containing a dicopper unit similar to the coordination geometry of the active sites of natural phosphatase and tyrosinase enzymes. This catalyst aided in phosphate ester bond hydrolysis and hydrogen peroxide decomposition, ultimately achieving high detoxification efficiency against both a nerve agent simulant (diethoxy-phosphoryl cyanide (DECP)) with a half-life of 3.5 min and a sulfur mustard simulant (2-chloroethyl ethyl sulfide (CEES)) with a half-life of 4.5 min, making it competitive with other reported materials. The dicopper sites in ZZU-282 provide versatile binding modes with the substrates, thereby promoting the activation of substrates and enhancing the catalytic efficiency. A combination of postmodified metal exchange control experiments, density functional theory calculations, and catalytic evaluations confirmed that dual Cu sites are the active centers promoting the catalytic reaction. This study offers a new design perspective to achieve advanced catalysts for CWA detoxification.

When Chemicals Go to the Dark Side

When Chemicals Go to the Dark Side

Unexpected Stability of a Prodrug to Enzymatic Hydrolysis within a Hydrated HPMC Matrix Tablet.

The uptake of alkaline phosphate present in dissolution medium into a hydrating hydroxypropyl methylcellulose matrix tablet and that its activity was retained therein was demonstrated. This presents a risk to the stability of prodrugs that are substrates of this enzyme such as phosphonooxymethyl derivative prodrugs. It was found that fostemsavir, a phosphonooxymethyl derivative prodrug being developed for the treatment of HIV-1 infection, was unexpectedly resistant to hydrolysis within a hydrated HPMC matrix when subjected to drug release testing in media containing alkaline phosphatase. Studies indicated that this was not due to microenvironmental pH effects, osmolality effects or effective phosphate concentration effects associated with the presence of the prodrug. That the prodrug and not its parent could affect enzyme activity in a concentration dependent manner, and that another phosphate ester prodrug fosphenytoin did not inhibit alkaline phosphatase activity within a hydrated HPMC matrix suggested that the unexpected stability of the HIV-1 therapy prodrug may be associated with the ability of the phosphate group-containing compound itself to inhibit the enzyme at the concentrations it exists at in the hydrated dosage form and so enables the development of the compound in this type of dosage form.

Predictive Tools to Assist the Development of Flame Retarded Phosphorus‐Free Polypropylene and Identify Synergy between Flame Retardants

AbstractThe development of flame‐retarded polypropylene is carried out with the ultimate goal of obtaining a UL94‐V0 not only halogen‐ but also phosphorus‐free formulation because phosphate rocks, precursors of phosphorus‐based flame retardants, are recognized as critical raw materials within Europe. Different flame retardants (FRs) (melamine, phosphate ester, and radical generator) are used. A statistical approach is applied to predict UL94 behavior and develop optimized formulations in terms of cost, filler or phosphorus content. Models for most common “nondripping” as well as “nonflaming dripping” UL94‐V0 behavior are proposed. The experimental validation has shown the reliability of the models proposed and their usefulness to assist and speed up the development of optimized formulations. The use of the models also gives insight into the interactions between flame retardants, thus suggesting if potential synergies take place, before studying the FRs system in details. Indeed, synergies between melamine and phosphate ester as well as melamine and radical generator are discovered. Thanks to these synergies that V0 rating can be achieved. A UL94‐V0 formulation, comprising melamine and radical generator, is identified as the optimal halogen‐ and phosphorus‐free formulation with a reduced filler content.

Functional Characterization of a HAD Phosphatase Involved in Capsular Polysaccharide Biosynthesis in Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium found in the intestinal tracts of chickens and many other farm animals. C. jejuni infection results in campylobacteriosis, which can cause nausea, diarrhea, fever, cramps, and death. The surface of the bacterium is coated with a thick layer of sugar known as the capsular polysaccharide. This highly modified polysaccharide contains an unusual d-glucuronamide moiety in serotypes HS:2 and HS:19. Previously, we have demonstrated that a phosphorylated glucuronamide intermediate is synthesized in C. jejuni NCTC 11168 (serotype HS:2) by cumulative reactions of three enzymes: Cj1441, Cj1436/Cj1437, and Cj1438. Cj1441 functions as a UDP-d-glucose dehydrogenase to make UDP-d-glucuronate; then Cj1436 or Cj1437 catalyzes the formation of ethanolamine phosphate or S-serinol phosphate, respectively, and finally Cj1438 catalyzes amide bond formation using d-glucuronate and either ethanolamine phosphate or S-serinol phosphate. Here, we investigated the final d-glucuronamide-modifying enzyme, Cj1435. Cj1435 was shown to catalyze the hydrolysis of the phosphate esters from either the d-glucuronamide of ethanolamine phosphate or S-serinol phosphate. Kinetic constants for a range of substrates were determined, and the stereoselectivity of the enzyme for the hydrolysis of glucuronamide of S-serinol phosphate was established using 31P nuclear magnetic resonance spectroscopy. A bioinformatic analysis of Cj1435 reveals it to be a member of the HAD phosphatase superfamily with a unique DXXE catalytic motif.

Lewis acid-catalyzed Pudovik reaction-phospha-Brook rearrangement sequence to access phosphoric esters.

Herein, we report a Lewis acid-catalyzed Pudovik reaction–phospha-Brook rearrangement sequence between diarylphosphonates or -phosphinates and α-pyridinealdehydes to access valuable phosphoric ester compounds. This transformation provides an extended substrate scope that is complementary to similar previously reported base-catalyzed transformations.

Surface modification of glass fibers for flame retardant and reinforced polyamide 6 composites

Glass fiber (GF) reinforced polyamide 6 (PA6) is a widely used composite. However, the GFs act as candlewick in burning, as a result, the PA6 continues burning after ignition. In this work, a novel flame retardant spirocyclic phosphate ester aminosilane (SPEA) was prepared, and it was self-crosslinked on the surface of the GF. The coated GF (SPEA-GF) was used with red phosphorus (RP) together in PA6. The flammability evaluation demonstrated that the PA6/SPEA-GF/RP (mass ratio of 64.5/30/5.5) composite passed the UL-94 V-0 grade in the vertical burning test, and the LOI value reached 30.5%. In the cone calorimeter test, the peak heat release rate (pHRR) of the PA6/GF (70/30) sample was 622 kW/m2; for the A6/SPEA-GF/RP sample, pHRR was sharply reduced to 356 kW/m2. Furthermore, the tensile, flexible, and impact strength of the PA6/SPEA-GF/RP composite were nearly equal to that of the PA6/GF (70/30) sample. This work provided a feasible solution to enhance the flame retardancy and mechanical performance of PA6/GF composites simultaneously.

A novel bio‐based, flame retardant and latent imidazole compound—Its synthesis and uses as curing agent for epoxy resins

AbstractComposite industry is constantly seeking multifunctional, high performance and environmentally friendly materials. Herein reported is a novel bio‐based, flame retardant and latent epoxy curing agent synthesized from wood vanillin, phosphate ester and imidazole. Experiment data from combustion test demonstrated that the cured epoxy resin with this new curing agent, designated as VAIP, rendered effective flame retardant property with limit of oxygen of 38.4%, UL‐94 V‐0 rating, total heat release of 48 MJm−2 and peak heat release rate of 435 KWm−2. Thermal mechanical tests showed its significantly higher char yield of 35% at 800°C and thermal stability with the temperature of initial weight loss rate of above 350°C, compared to reference epoxy resins cured by conventional cyano‐imidazole (CEMI). This new phosphorus containing imidazole curing agent also demonstrated potential application in fiber‐reinforced composite by rendering comparable Tg, for both dry and hot‐wet conditions, as well as higher interlaminar and shear strengths, when compared with the reference composite. Dynamic rheological isothermal measurement of the epoxy resin viscosity further concluded that VAIP had significantly better latency of 580 min at 60°C and 14 days at room temperature, resulting in much longer pot life or shelf life for the formulated resins and their prepreg.

Self-healing capability of novel eco-epoxy adhesives based on the modified tannic acid on Al adherends tested in a single lap joint

The aim of this paper is to study the self-healing capability of fractured Al joints bonded with novel eco-epoxide adhesives synthesized from a bio-renewable raw material (tannic acid – TA). Two synthesized eco-epoxy components based on TA, (A) glycidyl ether and (B) glycidyl phosphate ester of TA, were used as a replacement for the toxic epoxy component based on Bisphenol A. The effect of the eco-epoxy components on the self-healing capability was measured as a recovery of shear strength in a single lap joint (SLJ) test after complete failure, which was compared to the reference epoxy (R). The self-healing procedure was performed in an autoclave at 180 °C for 2 h and 2 bars. A combination of two monitoring techniques, Digital Image Correlation (DIC) and Acoustic Emission (AE), was used to monitor the strain distribution and damage propagation in the SLJ. The measured shear stress of A and B adhesives in the SLJ had values in the range of 2.3–5.1 MPa. A fracture analysis showed complete adhesive failure for all the tested adhesives, which was not affected by the self-healing process. Out of all adhesives, only the A adhesive demonstrated the capability to heal. The recovery of the shear strength for adhesive A was higher than 50% of the virgin case. In addition, the AE analysis managed to capture a clear distinction between the signals for the virgin and the self-healed tests for adhesive A. Results obtained in this study highlighted the promising potential of using bio-based epoxy adhesives in structural adhesive bonding with the possibility of using self-healing in the recovery of the strength of such bonded joints.

Dynamics of adhesive systems development in dental practice

BACKGROUND: Тhe article presents a review of adhesive systems in terms of their component chemical composition. Seven generations of adhesive systems have been studied. The first generation of adhesive systems emerged in the 1970s. The result of the action was the bond reaction of the adhesive with calcium enamel and dentin. Glycerophosphoric acid dimethacrylate was used. The use of methacrylates in adhesive systems was widespread because polymers with high biological indifference to biological objects are formed when they are polymerized in combination with acrylic resin. The second generation used a lubricated layer to obtain higher adhesion rates. Chloro-substituted phosphate esters of various monomers were used as active groups. The main compound mechanism remained the ionic binding of calcium dentine by chlorophosphate groups. The third generation used a lubricated layer to attach the composite to the dentin in the same way as the second generation. In the chemical composition, aluminosilicates, aluminitrates, hydroxyethylmethacrylate (HEMA), 4-methacryloxyethyltrimethyl anhydride (4-META), and other substances were most often used as active groups. The fourth generation is a multicomponent system that provides a three- and four-step application technique. These systems contain three to four components (conditioner, primer, and adhesive). The technique of their use includes three stages, namely, etching with 37% orthophosphoric acid, priming, and bonding. Adhesive systems of the fifth generation are two-component systems that provide a two-step technique of application. First, acid (etching) is applied to the tooth tissue, and second, the adhesive itself. Adhesive systems of the sixth and seventh generations are one-component self-etching since the adhesive contains acid. From a chemical point of view, these adhesive systems are a mixture of phosphoric esters and adhesive substances. Therefore, analyzing the adhesive composition of seven generations in such way, the mechanism of chemical interaction of adhesive components with hydroxyapatite and dentin has not significantly changed; however, the number of hydrophobic fragments has increased, which significantly increases dentin contact.

In vitro and in vivo pharmacological profile of OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan

Tolvaptan is an orally active vasopressin V2 receptor antagonist and used for the treatment of volume overload in some disease as an aquaretic. Tolvaptan sodium phosphate (OPC-61815) is a pro-drug of tolvaptan that was designed to improve water solubility and enable intravenous use. The conversion of OPC-61815 to tolvaptan was evaluated for in vitro and in vivo pharmacokinetic studies. The pharmacodynamics of OPC-61815 were evaluated for in vitro receptor binding affinity, in vivo aquaretic and anti-edematous action. The solubility of OPC-61815 in water at 25 °C was 72.4 mg/mL and more than 100,000 times the solubility of tolvaptan. OPC-61815 was hydrolyzed to tolvaptan by human tissue S9 fractions and main enzyme of hydrolysis was alkaline phosphatase. After intravenous administration of OPC-61815 to rats and dogs, tolvaptan was detected in plasma within 5 min and the bioavailability of tolvaptan was 57.7% and 50.9%, respectively. Binding affinity of OPC-61815 for the human V2 receptor was 1/14 weaker than that of tolvaptan. OPC-61815 exerted dose-dependent aquaretic action in rats and dogs and a corresponding anti-edematous action in rat edema models. These results suggest that OPC-61815, a water-soluble phosphate ester pro-drug of tolvaptan, is an effective aquaretic by converting to tolvaptan after intravenous administration.

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass

Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.