Development and validation of the spectrophotometric method of butaphosphan determination in veterinary preparations.

A simple and sensitive method has been developed for the determination of butaphosphan in single- and multi-component veterinary preparations. The method is based on the combustion of organic matter prior to the formation of phosphate and the subsequent formation of the phosphoric­molybdenum complex in the presence of molybdate and ascorbic acid as a reducing agent in the sulfate acid medium. The intensity of light absorption of the colored analytical form of "molybdenum blue" is measured at a wavelength of 830 nm in the range of 0.3-9.0 µg/ml of butaphosphan, where Beer's law is obeyed, has been measured. The reaction conditions and other experimental parameters influencing the reaction transition and the stability of the colored complex have been thoroughly investigated and optimized for the quantitative determination of butaphosphan. Validation of the developed method according to the requirements has been carried out State Pharmacopoeia of Ukraine garmonised with European Pharmacopoeia and its suitability for the analysis of the selected veterinary preparations for the quantitative content of butaphosfan has been confirmed. The results of the determination of butaphosphan in six veterinary preparations of domestic and imported products have been obtained with the help of the developed method. The received results are in agreement with the results obtained by the method of potentiometric titration.

A modified quick, easy, cheap, effective, rugged, and safe cleanup method followed by liquid chromatography-tandem mass spectrometry for the rapid analysis of perchlorate, bromate and hypophosphite in flour.

A selective, sensitive and useful method, based on modified QuEChERS cleanup combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the negative-ion electrospray ionization (ESI-) mode, was developed and validated for the simultaneous determination of three inorganic anions including perchlorate (ClO4-), bromate (BrO3-) and hypophosphite (H2PO2-) in flour. The extraction parameters and LC-MS/MS conditions were optimized by single-factor experiment and sorbent combination in modified QuEChERS clean-up was optimized through response surface analysis. Three target analytes were separated on a normal-phase Phenomenex Luna Silica (2) column (150mm×2.0mm, 5µm, 100Å) with the mobile phase of a mixture of 5mmol/L ammonium acetate water solution and acetonitrile, detected by MS/MS under multiple reaction monitoring and quantified by external standard method. The developed method was validated in terms of the sensitivity, linearity, accuracy and precision, and matrix effect. The method showed a good linearity (R2>0.999) for all analytes in their respective concentration ranges. The ILOQs and MLOQs for perchlorate, bromate and hypophosphite were 0.1, 0.5, 5.0µg/L and 2.0, 6.0, 60.0µg/L, respectively. The average recoveries of three target analytes from the negative samples spiked at three different concentrations were in a range from 84.6% to 104.9%. The intra-day precision (n=6) and inter-day precision (n=5) of the target analytes were in the ranges of 2.9%-6.9% and 6.4%-8.2%. The matrix effect of this method was observed between 0.83 and 1.17 and was acceptable. The validated method was successfully applied to determine the concentrations of these inorganic anions in flour. Results found that perchlorate and hypophosphite were detected in 33 out of 50 and 7 out of 50 flour samples.

Analysis of toldimfos in porcine muscle and bovine milk using liquid chromatography-triple quadrupole mass spectrometry.

An analytical method was developed for the detection of toldimfos sodium residues in porcine muscle and bovine milk using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) analysis. The drug was extracted from muscle and milk using 10 mm ammonium formate in acetonitrile and then purified using n-hexane. The drug was well separated on a Luna C18 column using a mixture of 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B) as the mobile phase. Good linearity was achieved over the tested concentration range (0.005-0.03 mg/kg) in matrix-matched standard calibration. The determination coefficients (R2 ) were 0.9942 and 0.9898 for muscle and milk, respectively. Fortified porcine muscle and bovine milk contained concentrations equivalent to and twice the limit of quantification (0.005 mg/kg) yielded recoveries in the range of 75.58-89.74% and relative standard deviations of ≤8.87%. Samples collected from large markets located in Seoul, Republic of Korea, tested negative for toldimfos sodium residue. In conclusion, ammonium formate in acetonitrile can effectively extract toldimfos sodium from porcine muscle and bovine milk without solid-phase extraction, which is usually required for cleanup before analysis. This method can be applied for the routine analysis of toldimfos in foods of animal origins.

Comparative assessment of the environmental hazards of and exposure to perfluoroalkyl phosphonic and phosphinic acids (PFPAs and PFPiAs): Current knowledge, gaps, challenges and research needs.

Perfluoroalkyl phosphonic and phosphinic acids (PFPAs and PFPiAs) are sub-groups of per- and polyfluoroalkyl substances (PFASs) that have been commercialized since the 1970s, particularly as defoamers in pesticide formulations and wetting agents in consumer products. Recently, C4/C4 PFPiA and its derivatives have been presented as alternatives to long-chain PFASs in certain applications. In this study, we systematically assess the publicly available information on the hazardous properties, occurrence, and exposure routes of PFPAs and PFPiAs, and make comparisons to the corresponding properties of their better-known carboxylic and sulfonic acid analogs (i.e. PFCAs and PFSAs). This comparative assessment indicates that [i] PFPAs likely have high persistence and long-range transport potential; [ii] PFPiAs may transform to PFPAs (and possibly PFCAs) in the environment and biota; [iii] certain PFPAs and PFPiAs can only be slowly eliminated from rainbow trout and rats, similarly to long-chain PFCAs and PFSAs; [iv] PFPAs and PFPiAs have modes-of-action that are both similar to, and different from, those of PFCAs and PFSAs; and [v] the measured levels of PFPAs/PFPiAs in the global environment and biota appear to be low in comparison to PFCAs and PFSAs, suggesting, for the time being, low risks from PFPAs and PFPiAs alone. Although risks from individual PFPAs/PFPiAs are currently low, their ongoing production and use and high persistence will lead to increasing exposure and risks over time. Furthermore, simultaneous exposure to PFPAs, PFPiAs and other PFASs may result in additive effects necessitating cumulative risk assessments. To facilitate effective future research, we highlight possible strategies to overcome sampling and analytical challenges.

Highly sensitive determination of dialkyl phosphinate acids in environmental samples by ion chromatography tandem mass spectrometry.

Dialkyl phosphinate acids (DPAs) are the hydrolysates of aluminum dialkyl phosphinates (ADPs), one class of emerging phosphorus flame retardants since brominated flame retardants have been gradually phased out in recent years. It has been found that once dissolved in water, ADPs are completely hydrolyzed and exist as DPAs. However, there is no report on the determination of DPAs in environmental water samples. For the first time, we developed a method for the analysis of trace DPAs and ADPs in different environmental samples, including waters, soils and sediments. In this proposed method, MAX cartridges were employed for the purification, and ion chromatography (IC) tandem mass spectrometry (MS) method with large volume injection (200µL) and postcolumn addition of methanol and NH3·H2O were employed for the determination of DPAs and ADPs. The matrix effects were <16% for water samples and <25% for soil/sediment samples, which were greatly improved in comparison to the liquid chromatography (LC) tandem MS determination. Determined at three fortified levels of 0.02µg/L, 0.2µg/L and 1.0µg/L, the mean recoveries were from 75.8% to 110.2%, with an acceptable coefficient of variation (3.3-20%, n=6) for water samples. The limits of the method were 3.5-9.3ng/L for DPAs in environmental water samples, and 0.06-0.09µg/kg for DPAs and ADPs in soil and sediment samples. For soil and sediment samples, results determined by the present IC-MS method were in good agreement with that determined by LC-MS in our previous study.

A small structural change resulting in improved properties for product development.

AZD9343 is a water-soluble gamma amino butyric acid (GABAB) agonist intended for symptomatic relief in gastroesophageal reflux disease (GERD) patients. The compound has good chemical stability in aqueous solutions, as well as in the solid state. Only one crystal modification has been observed to date. This polymorph is slightly hygroscopic (1.5% water uptake at 80% relative humidity (RH)), which is an improvement compared to the structurally similar agonist lesogaberan (AZD3355) which liquefies at 65% RH. Since the substance is very polar and lacks a UV chromophore, conventional separation and detection techniques cannot be used to characterize the substance and its impurities. The analytical techniques are described, focusing on the capillary electrophoresis method with indirect UV detection for assay and purity, the liquid chromatographic method for enantiomeric separation with derivatization with UV chromophore and three complementary nuclear magnetic resonance (NMR) approaches ((31)P-NMR, (13)C-NMR and (1)H-NMR) for impurities. For oral solutions, it was important to select the right concentration of phosphate buffer for the specific drug concentration and routinely use small additions of EDTA. I.V. solutions containing physiological saline as tonicity modifier could not be stored frozen at -20 °C. Properties of AZD9343 will be discussed in light of experiences from the structurally similar lesogaberan and (2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA).

Aluminum dialkyl phosphinate flame retardants and their hydrolysates: analytical method and occurrence in soil and sediment samples from a manufacturing site.

Aluminum dialkyl phosphinates (ADPs) are emerging phosphorus flame retardants due to their superior characteristics, but their analytical method, and occurrence and fate in environments have never been reported. For the first time, we developed a method for the analysis of trace ADPs and their hydrolysates (dialkyl phosphinic acids, DPAs), and studied their occurrences and fates in soils and sediments. We found that ADPs are hardly dissolved in water and organic solvents, but are dissolved and hydrolyzed to DPAs in 30 mM NH3·H2O, thus both ADPs and DPAs can be determined by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the form of DPAs. ADPs and DPAs in soil and sediment samples were determined by (i) extracting both ADPs and DPAs with 75 mM NH3 · H2O, and selectively extract DPAs only with formic acid-water-methanol (5:5:90, v/v/v); (ii) quantifying the total content of ADPs and DPAs, and DPAs by LC-MS/MS analysis of the DPA contents in the former and the latter extract, respectively; and (iii) calculating ADPs from the content difference between the former and the latter extracts. The limit of quantifications (LOQs) of the proposed method were 0.9-1.0 µg/kg, and the mean recoveries ranged from 69.0% to 112.4% with relative standard deviations ≤ 21% (n = 6). In soil and sediment samples around a manufacturing plant, ADPs and DPAs were detected in surface soils in the ranges of 3.9-1279.3 and 1.0-448.6 µg/kg, respectively. While ADPs were found in all the samples of the soil and sediment cores from the drain outlet and the waste residue treatment site at levels ranging from 30.8 to 4628.0 µg/kg, DPAs were found in more than 90% of these samples with concentrations in the range of 1.1-374.6 µg/kg. The occurrences of ADPs and DPAs are not in correlation with the total organic carbon, whereas the occurrences of DPAs are highly correlated with the sample pH. Our study also suggests that the DPAs in the samples sourced from the hydrolysis of ADPs. The high hydrolysis degrees of ADPs (up to 49.6%) suggest that once released into the environment, ADPs are likely to coexist with their hydrolysates. Thus, to evaluate the environmental safety of ADPs, the environmental behavior and toxicity of both ADPs and DPAs should be considered.

Development of analytical methods for the quantification of metabolites of lesogaberan in a MIST investigation.

Analytical methods were developed for the determination of six metabolites of lesogaberan to be used in quantitative determinations of metabolites according to the guidelines of Metabolites in Safety Testing. The γ-amino butyric acid type B receptor agonist lesogaberan and its metabolites are small polar molecules and hydrophilic interaction liquid chromatography was found to be a suitable separation mode. The samples were prepared using protein precipitation and negative electrospray ionization tandem mass spectrometry was used for detection. Initially, exploratory methods for six metabolites were set up for analysis of human plasma samples taken after repeated administration of a high oral dose of lesogaberan. The purpose was to establish which metabolites were present at concentrations significant for further investigation. Four of the six metabolites were then found at clearly detectable concentrations. The analytical methods for these four metabolites were further elaborated and then taken through a qualification procedure, which showed acceptable accuracy (86-114%), precision (<9%) and good linearity in the range 0.03-5 µmol/L. No interferences were seen from endogenous plasma components.

Early pharmaceutical evaluation of a crystalline and hygroscopic GABAB receptor agonist.

Lesogaberan is a potent gamma amino butyric acid agonist and has been evaluated for its utility in treatment of gastroesophageal reflux disease. Lesogaberan is a crystalline substance that absorbs considerable amounts of water above 65% relative humidity (RH) where it also liquifies. As a result of the hygroscopicity of the zwitterionic form an investigation of different salt forms was performed. Since the test compound is polar and lacks ultraviolet (UV) chromophore, conventional separation and detection techniques could not be used to characterise the test compound and the impurities. The analytical techniques are described, focusing on the capillary electrophoresis method with indirect UV detection for purity, the liquid chromatographic method for enantiomeric separation with derivatisation with UV chromophore and two complementary nuclear magnetic resonance (NMR) approaches (¹9F-NMR and ¹H-NMR) for impurities. The stability study in solution showed that solutions between pH 5 and 7 were the most stable ones, but after some time degradation occurred at room temperature. When bulk lesogaberan was stored at 25°C/60% RH no chemical degradation was observed after 1 year. At 40°C/75% RH, where the compound liquefies, a significant degradation was observed after 1 month. However, in a closed container (= 40°C) or as a napsylate salt, no degradation of lesogaberan was observed at 40°C/75% RH.

Facile synthesis of hybrid sulfonophosphinodipeptides composing of taurines and 1-aminoalkylphosphinic acids.

Both sulfonopeptides and phosphonopeptides are important analogs of naturally occurring peptides and have been widely used as enzyme inhibitors and haptens for producing catalytic antibodies due to their tetrahedrally structural features. A series of hybrid sulfonophosphinodipeptides composing of taurines and 1-aminoalkylphosphinic acids were first and conveniently synthesized in satisfactory to good yields via a Mannich-type reaction of N-benzyloxycarbonylaminoalkanesulfonamides, aldehydes, and aryldichlorophosphines, and subsequent hydrolysis. The current method provides an efficient and direct synthesis of hybrid sulfonophosphinodipeptides.

Design, synthesis, and metal binding of novel Pseudo- oligopeptides containing two phosphinic acid groups.

Phosphinic compounds have potential as amide-bond mimetics in the development of novel peptidomimetics, enzyme inhibitors, and metal-binding ligands. Novel pseudo-oligopeptides with two phosphinic acid groups embedded in the peptide backbone serving as amide-bond surrogates, Psi[P(O,OH)--CH(2)], were targeted. A series of linear and cyclic pseudo-oligopeptides with two phosphinic acid groups arrayed at different positions in the peptide sequence were designed, including Ac--Phe--{(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly}(2)--NH(2) (P2), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P3), Ac--NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe--(R,S) --AlaPsi[P(O,OH)--CH(2)]Gly--NH(2) (P4), cyclo{NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe}(2) (P5), and cyclo[NH--(R,S)--AlaPsi[P(O,OH)--CH(2)]Gly--Phe--Phe](2) (P6). They were synthesized via conventional Fmoc chemistry on solid support utilizing Fmoc-protected phosphinic acid-containing pseudo-dipeptide fragment, i.e. Fmoc--(R,S)--AlaPsi[P(O,OCH(3))--CH(2)]Gly--OH. The pseudo-peptides containing two phosphinic acid groups exhibited the highest binding affinity and selectivity for Fe(III) among the 10-metal ions screened by ESI-MS analysis--Cu(II), Zn(II), Co(II), Ni(II), Mn(II), Fe(II), Fe(III), Al(III), Ga(III), and Gd(III). P4 and P6 with 11-atom linkages between the two phosphinic acids preferred intramolecular metal binding to form 1:1 ligand/metal complexes. As revealed by competition experiments, P4 showed the highest relative binding affinity among the six compounds tested. Noteworthy, P4 also showed higher relative binding affinity than similar dihydroxamate-containing pseudo-peptides reported previously. The novel structural prototype and facile synthesis along with selective and potent Fe(III) binding strongly suggest that pseudo-peptides containing the two or more phosphinic groups as amide-bond surrogates deserve further exploration in medicinal chemistry.

Determination of impurities in flame retardant monomer 2-carboxyl ethyl(phenyl) phosphinic acid by ion chromotography.

In this paper, a method based on ion chromatography (IC) with conductivity detector was developed for the determination of impurities including phenyl phosphinic acid (BPA), phenyl phosphonic acid (PPOA) and crylic acid in flame retardant monomer 2-carboxyl ethyl(phenyl) phosphinic acid (CEPPA). Under favorable chromatographic conditions, good linear relationship, sensitivity and reproducibility were obtained. Detection limits of BPA, PPOA and crylic acid were 1.5, 0.5, 0.4 microg l(-1), respectively. Relative standard deviations (RSD) of repeated analyses were less than 2.22% (n=10). The real samples (white crystal) have been tested and rate of recovery were 89-108%. It was confirmed that this method could be used in the analysis of flame retardant monomers.

Detection of phosphorus oxyanions in synthetic geothermal water using ion chromatography-mass spectrometry techniques.

Recent developments in microbiology suggest that reduced inorganic phosphorus oxyanions, including hypophosphite and phosphite, may be present in nature. These studies have inspired the development of specific and sensitive methods that detect phosphorus oxyanions in natural water. This paper will discuss a new technique that couples suppressed conductivity ion chromatography (Dionex AS17 analytical column and potassium hydroxide eluent) with electrospray mass spectrometry (IC/MS) with limits of detection nearly 200 times lower than those reported using suppressed conductivity detection. The technique was optimized for the detection of hypophosphite, phosphite, and phosphate in a synthetic geothermal water matrix. Samples were pre-treated with silver and sulfonic acid cartridges, and injection loop sizes as large as 800microl were employed to enhance instrument sensitivity. All peaks were clearly resolved, and calibrations were linear with estimated 3sigma limits of detection of 0.011, 0.0020, and 0.029microM for hypophosphite, phosphite, and phosphate, respectively.

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.

Steroid phosphorus compounds. V--Mass spectrometry of phosphinic esters of monohydroxy steroids.

The mass spectra of the dimethylphosphinic, dimethylthiophosphinic and dimethylphosphinous ester derivatives of several monohydroxy steroids are reported. The fragmentations of the derivatized steroids largely depend on the nature of the phosphorus-containing ester group. Phenolic ester derivatives exhibit the base peak at the molecular ion, whereas the spectra of the secondary phosphinic esters are dominated by very intense protonated phosphinic acid ions [Me2P(XH)(OH)]+ at m/e 95 (X =O) or at m/e 111 (x = s). The present results also indicate the low ionization potential for the phosphinic ester group. Due to their good gas chromatographic properties, these steroid derivatives appear to be particularly suitable for gas chromatographic mass spectrometric analysis of biochemical materials.