Development and evaluation of a novelty single-step cleanup followed by HPLC-QTRAP-MS/MS for rapid analysis of tricaine, tetracaine, and bupivacaine in fish samples.

A novelty single-step cleanup method combined with HPLC coupled with triple quadrupole-linear ion trap MS/MS (HPLC-QTRAP-MS/MS) was developed for the analysis of tricaine, tetracaine, and bupivacaine in fish tissue. The target analytes were extracted using acetonitrile based on the modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method under ultrasound irradiation. A cheap analytical filtration syringe (CAFS) cleanup column for single-step purification was proposed first; 300 mg of primary/secondary amino was proposed as the optimum purification sorbent; 1 mL of acetonitrile extract was transferred into a CAFS cleanup column and purified for analysis using HPLC-QTRAP-MS/MS. The limits of detection and the limits of quantification were 2.0 and 5.0 µg kg-1 , respectively. The recoveries were in the range of 88.73-108.72%. Inter-day and intra-day relative standard deviations were lower than 15% for all analytes. The developed method has been applied to measure real samples obtained from the local market.

Stable isotope dilution assay for the accurate determination of tricaine in fish samples by HPLC-MS-MS.

Tricaine methanesulfonate is one of most commonly used anesthetics in fish during blood sampling, artificial propagation and long-distance transportation. In this study, an accurate method for the quantitative determination of tricaine in fish samples by a stable isotope dilution assay coupled with high-performance liquid chromatography-triple quadrupole mass spectrometry was developed. Tricaine-D5 was synthesized and used as an isotopically labeled internal standard for the determination of tricaine. The analytical performance of the method was validated for tricaine determination in marine fish and freshwater fish. The determination of tricaine was linear in the range of 2.0-200.0 µg L-1 . The limit of detection and limit of quantitation for fish muscle tissues were 1.0 and 4.0 µg kg-1 , respectively. Good recoveries were obtained in the range of 92.08-97.50%. The inter- and intra-assay relative standard deviations (RSD values) were investigated, and the values were 0.39-3.01 and 0.85-2.77%, respectively. The values of CCα and CCß were 10.21-10.43 and 10.42-10.87 µg kg-1 , respectively. The clearance of MS-222 from grass carp was further studied using our method. The results demonstrate that MS-222 could be well absorbed and rapidly eliminated after bath administration.

Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction.

An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 µM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.

Antibacterial sulfur-containing platensimycin and platencin congeners from Streptomyces platensis SB12029.

The platensimycin (PTM) and platencin (PTN) class of natural products are promising drug leads that target bacterial and mammalian fatty acid synthases. Natural congeners and synthetic analogues of PTM and PTN have been instrumental in determining their structure-activity relationships, with only a few analogues retaining the potencies of PTM and PTN. Here we describe the identification and isolation of two new sulfur-containing PTM congeners (3 and 5) from the engineered dual PTM-PTN overproducing Streptomyces platensis SB12029. Structure elucidation of platensimycin D1 (5), a sulfur-containing PTM pseudo-dimer, revealed the existence of its presumptive thioacid precursor (3). The unstable thioacid 3 was isolated and confirmed by structural characterization of its permethylated product (6). LC-MS analysis of crude extracts of SB12029 confirmed the presence of the thioacid analogue of PTN (4). The minimum inhibitory concentration (MIC) was determined for 5 revealing retention of the strong antibacterial activity of PTM.

Comparison of pulse glow discharge-ion mobility spectrometry and liquid chromatography with tandem mass spectrometry based on multiplug filtration cleanup for the analysis of tricaine mesylate residues in fish and water.

Analytical methods based on multiplug filtration cleanup coupled with pulse glow discharge-ion mobility spectrometry and liquid chromatography tandem mass spectrometry were developed for the analysis of tricaine mesylate residue in fish and fish-raising water samples. A silica fiber holder and an appropriate new interface were designed to make the direct introduction of the fiber into the pulse glow discharge-ion mobility spectrometry introduction mechanism. The multiplug filtration cleanup method with adsorption mixtures was optimized for the determination of tricaine mesylate in fish samples. Good linear relationships were obtained by the two methods. For fish samples, limits of detection were 6 and 0.6 µg/kg by ion mobility spectrometry and liquid chromatography with tandem mass spectrometry, respectively. The matrix effect of the established liquid chromatography tandem mass spectrometry method was negligible for fish samples but that of the ion mobility spectrometry method was not. The two methods were compared. The ion mobility spectrometry system could be used a rapid screening tool on site with the advantage of rapidity, simplicity, and portability, and the liquid chromatography tandem mass spectrometry system could be used for validation in laboratory conditions with the advantage of lower limit of detection, stability, and precision.

Analysis of persistent contaminants and personal care products by dispersive liquid-liquid microextraction using hydrophobic magnetic deep eutectic solvents.

In this work, hydrophobic magnetic deep eutectic solvents (HMDESs) were used in the development of a simple and rapid dispersive liquid-liquid microextraction (DLLME) approach coupled to high performance liquid chromatography with UV detection (HPLC-UV) for the determination of ten organic contaminants including five polycyclic aromatic hydrocarbons, four UV filters, and a pesticide from water at trace levels. The HMDESs were prepared by mixing a hydrogen bond acceptor, metal halide salt, and hydrogen bond donor in suitable molar ratios. Two HMDESs, 2 tetraoctylammonium bromide ([N8888+][Br-]): cobalt chloride (CoCl2): 4 octanoic acid (OA) and 3 trioctylphosphine oxide (TOPO): neodymium chloride (NdCl3): 3 OA, offered the highest analyte extraction efficiency overall and were chosen as suitable solvents for validation of the microextraction method. Under optimized extraction conditions, the method required 30 µL of HMDES as extraction solvent, acetone (87.5 µL) as disperser solvent, a NaCl concentration of 30% (w/v), and an extraction time of 120 s at 20°C. Enrichment factors of the analytes ranged from 44.6 for 3-(4-methylbenzylindene) camphor to 66.0 for 2-ethylhexyl-4-(dimethyl)aminobenzoate. The method provided low limits of detection (LODs) ranging from 0.5 to 4.5 µg L-1, and acceptable precision, with RSD values lower than 9.6%. Furthermore, the validated method was successfully applied for tap and lake water analysis, resulting in relative recoveries of spiked samples ranging between 94.7 and 119.2%.

Taxonomic profiling and functional gene annotation of microbial communities in sediment of river Ganga at Kanpur, India: insights from whole-genome metagenomics study.

The perennial river Ganga is recognized as one of India's largest rivers of India, but due to continuous anthropogenic activities, the river's ecosystem is under threat. Next-generation sequencing technology has transformed metagenomics in the exploration of microbiome and their imperative function in diverse aquatic ecosystems. In this study, we have uncovered the structure of community microbiome and their functions in sediments of river Ganga at Kanpur, India, at three polluted stretches through a high-resolution metagenomics approach using Illumina HiSeq 2500. Among the microbes, bacteria dominate more than 82% in the three polluted sediment samples of river Ganga. Pseudomonadota (alpha, beta, and gamma) is the major phylum of bacteria that dominates in three sediment samples. Genes involved in degradation of xenobiotic compounds involving nitrotoluene, benzoate, aminobenzoate, chlorocyclohexane, and chlorobenzene were significantly enriched in the microbiome of polluted stretches. Pathway analysis using KEGG database revealed a higher abundance of genes involved in energy metabolism such as oxidative phosphorylation, nitrogen, methane, sulfur, and carbon fixation pathways in the sediment metagenome data from the river Ganga. A higher abundance of pollutant degrading enzymes like 4-hydroxybenzoate 3-monooxygenase, catalase-peroxidase, and altronate hydrolase in the polluted microbiome indicates their role in degradation of plastics and dyes. Overall, our study has provided bacterial diversity and their dynamics in community structure and function from polluted river microbiome, which is expected to open up better avenues for exploration of novel functional genes/enzymes with potential application in health and bioremediation.

Determination of tricaine residues in fish by liquid chromatography.

Tricaine methanesulfonate (MS-222) is approved by the U.S. Food and Drug Administration, Center for Veterinary Medicine (CVM), as an anesthetic drug for select aquaculture species. It was approved for use as a handling aid with a 3 week withdrawal time. The drug is rapidly metabolized and excreted; therefore, CVM approved its use without requiring a regulatory method for drug residues in tissues. However, there are concerns that the drug may be used to sedate fish during transport to slaughter. A regulatory method will enable monitoring for unsafe residues of this drug resulting from extralabel use. We present a quantitative method, using LC at a target level of 0.1 mg/kg (ppm), for three different farmed species: salmon (Salmo salar); tilapia (Oreochromis spp.); and catfish (Ictalurus punctatus). The assay begins with an acetonitrile extraction, followed by filtration and mixed-mode cation-exchange solid-phase extraction cleanup. The extracts are analyzed by reversed-phase LC with UV detection at 320 nm. The method was validated by using fish fillets with incurred residues, control fish fillets, and fish fillets fortified at half the target level, the target level, and twice the target level (0.05, 0.1, and 0.2 ppm, respectively). For all species, accuracy is > or =80% and the RSD is < or =10%. The method complies with CVM performance criteria for the determination of veterinary drug residues.

Removal of tricaine methanesulfonate from aquaculture wastewater by adsorption onto pyrolysed paper mill sludge.

Tricaine methanesulfonate (MS-222) has been widely used in intensive aquaculture systems to control stress during handling and confinement operations. This compound is dissolved in the water tanks and, once it is present in the Recirculating Aquaculture Systems (RASs), MS-222 can reach the environment by the discharge of contaminated effluents. The present work proposes the implementation of the adsorption process in the RASs, using pyrolysed biological paper mill sludge as adsorbent, to remove MS-222 from aquaculture wastewater. Adsorption experiments were performed under extreme operating conditions, simulating those corresponding to different farmed fish species: temperature (from 8 to 30 °C), salinity (from 0.8 to 35‰) and different contents of organic and inorganic matter in the aquaculture wastewater. Furthermore, the MS-222 adsorption from a real aquaculture effluent was compared with that from ultrapure water. Under the studied conditions, the performance of the produced adsorbent remained mostly the same, removing satisfactorily MS-222 from water. Therefore, it may be concluded that the produced adsorbent can be employed in intensive aquaculture wastewater treatment with the same performance independently of the farmed fish species.

Determination of MS-222 in Water Samples by Solid-phase Extraction Coupled with Liquid Chromatography/Tandem Mass Spectrometry.

A practical solid-phase extraction (SPE) method coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed for the determination of the fish anesthetic MS-222 in water. Water samples were concentrated and purified using three SPE cartridges of different specifications. Elution curves of MS-222 were constructed using various methanol-water solutions on the different cartridges, and SPE conditions were optimized in accordance with the elution curves. The mobile phase containing methanol and 0.1% formic acid solution with a linear gradient elution was utilized to separate MS-222 on a C18 column. Detection was carried out by a triple-quadrupole mass spectrometry with an electrospray ion source in positive mode. Recoveries of three MS-222 fortified levels of 0.05, 0.5 and 5 µg/L ranged of 82.6-101% with relative standard deviations (RSDs) below 9.36%. The limit of detection (LOD) and limit of quantification (LOQ) of MS-222 were 0.01 µg/L and 0.03 µg/L, respectively. This method was satisfactorily applied to the determination of MS-222 in actual water samples collected from aquatic product transportation vehicles or from the natural water catchments.

Simultaneous determination of p-aminobenzoic acid, acetyl-p-aminobenzoic acid and p-aminohippuric acid in serum and urine by capillary gas chromatography with use of a nitrogen-phosphorus detector.

In various studies during recent years, the use of p-aminobenzoic acid has been described in screening tests for exocrine pancreatic function. A synthetic three-unit compound N-benzoyl-L-tyrosyl-p-aminobenzoic acid has been administered orally and hydrolysed in the small intestine in the presence of chymotrypsin to N-benzoyl-L-tyrosine and p-aminobenzoic acid. This study describes a convenient procedure in which, after a selective extraction and derivatization with diazomethane, capillary gas chromatography is used combined with nitrogen-sensitive detection. With the proposed procedure, p-aminobenzoic acid and its major metabolites, acetyl-p-aminobenzoic acid and p-aminohippuric acid, can be monitored in serum and in urine samples.

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.

Spectroscopic approach to estimation of microequilibrium constants of prototropic reactions of aminobenzoic acids.

The microequilibrium constants of protolytic dissociation of diprotic acids, dihydric bases, or ampholytes such as the aminobenzoic acids, with dissimilar ionizing groups, can be estimated by spectrophotometric titration and measurement of the molar absorptivity at the long wavelength absorption maximum of simple alkylated derivatives. The method is applicable when the long wavelength absorption spectral bands of the tautomeric species are well resolved. Compared to the traditional method of estimating microequilibrium constants using the dissociation constants of alkylated derivatives, the proposed method is simpler, faster, and more accurate.

Simultaneous determination of tetracaine and its degradation product, p-n-butylaminobenzoic acid, by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of tetracaine hydrochloride and its hydrolytic degradation product, p-n-butylaminobenzoic acid. Separation was achieved using a mu Bondapak C18 column and the eluent, water-acetonitrile-methanol (60:20:20), containing 0.06% (v/v) sulfuric acid, 0.5% (w/v) sodium sulfate, and 0.02% (w/v) sodium heptanesulfonate, at a flow rate of 2 ml/min Salicylic acid and propiophenone were used as internal standards. The UV detector response at 305 nm was linear for tetracaine hydrochloride in the 0.4--2.0-mg/ml range and for p-n-butylaminobenzoic acid in the 0.003--0.02-mg/ml range. The method is simple and precise.

High-performance liquid chromatographic assay for benzocaine and p-aminobenzoic acid including preliminary stability data.

A high-performance liquid chromatographic assay was developed that separates and quantitates benzocaine and its primary degradation product, p-aminobenzoic acid. This method is rapid, sensitive, and specific. Preliminary stability data obtained with this method demonstrate its utility for this purpose.

Controlled drug permeation I: controlled release of butamben through silicone membrane by complexation.

The effects of caffeine, beta-cyclodextrin, and povidone on the permeation behavior of butamben from saturated solutions in these complexing agents through a dimethyl polysiloxane membrane were investigated at 30 degrees. In all systems, these agents increased the rate of release over the plain saturated drug solution. The effect was more pronounced with caffeine and beta-cyclodextrin than with povidone. Interpretation of these results with the aid of solubility data for the corresponding systems led to the following generalization. For a fixed total (free and complexed) amount of drug available for release, sustained release is associated with systems containing more stable complexes. The practical value of this approach to the controlled release of drug is discussed.

Non-aqueous capillary electrophoresis of drugs: properties and application of selected solvents.

The electrophoretic mobility of selected acidic and basic test solutes have been determined in non-aqueous media prepared by adding various combinations of ammonium acetate, sodium acetate, methane sulphonic acid and acetic acid to acetonitrile, propylene carbonate, methanol, formamide, N-methylformamide, N,N-dimethylformamide and dimethylsulphoxide, respectively. The apparent pH (pH*) of these non-aqueous media have been measured and it was found that pH* is an important factor for the separations in non-aqueous capillary electrophoresis. However, in some solvents the concentration of sodium acetate has a strong influence on the mobility despite very small changes in pH*. Due to the fact that a change in one parameter influences a number of other parameters it is very difficult to conduct systematic studies in non-aqueous media and to compare the migration of the species at fixed pH* values from one solvent to another. Thus pH* is only of value for comparison when used with a specific solvent or solvent mixture. The viscosity of the above-mentioned solvents were measured at various temperatures and means to adjust the viscosity of the non-aqueous media used for capillary electrophoresis are discussed and the separation of ibuprofen and its major metabolites in urine is used as an example.

Application of stripping voltammetry to trace lead analysis in intermediates and final products of syntheses of pharmaceuticals.

Applications of differential pulse anodic stripping voltammetry using a new pen-type renewed hanging mercury electrode have been investigated for trace analysis of lead in pharmaceutical substances and intermediates of their syntheses, such as procaine hydrochloride, 4-aminobenzoic acid, methyl 4-aminobenzoate, 2-(4-chlor-3-aminobenzoyl) benzoic acid, benzyl 2-naphthyl ether, 5-aminoisophthalic acid, 3-aminobenzoic acid, 5-hydroxyisophthalic acid, and N, N'-dibenzylethylenediamine diacetate. Samples were dissolved in 1 M HCI or 1 M NaOH and the electrochemical scan was carried out. No sample mineralization was necessary. The method showed a good linearity up to 50-100 ppm Pb with a detection limit less than 100 ppb. The results agreed well, but were more precise than those obtained by atomic absorption spectrometry using air/acetylene flame atomisation.

The solubilization of some local anaesthetic ester of p-aminobenzoic acid by lysophosphatidylcholine.

The solubilization by lysophosphatidylcholine (LPC) of three n-alkyl esters of p-aminobenzoic acid has been studied. These esters have a local anaesthetic action. Quantitative studies show that the amount of compound solubilized is proportional to the LPC concentration and that solubilization increases in the order ethyl, n-propyl and n-butyl ester. 100MHz nmr studies indicate that the local anaesthetic esters are solubilized in the hydrocarbon interior of the LPC.

Purity and adulterant analysis of crack seizures in Brazil.

Cocaine represents a serious problem to society. Smoked cocaine is very addictive and it is frequently associated with violence and health issues. Knowledge of the purity and adulterants present in seized cocaine, as well as variations in drug characteristics are useful to identify drug source and estimate health impact. No data are available regarding smoked cocaine composition in most countries, and the smoked form is increasing in the Brazilian market. The purpose of the present study is to contribute to the current knowledge on the status of crack cocaine seized samples on the illicit market by the police of São Paulo. Thus, 404 samples obtained from street seizures conducted by the police were examined. The specimens were macroscopically characterized by color, form, odor, purity, and adulterant type, as well as smoke composition. Samples were screened for cocaine using modified Scott test and thin-layer chromatographic (TLC) technique. Analyses of purity and adulterants were performed with gas chromatography equipped with flame ionization detector (GC-FID). Additionally, smoke composition was analyzed by GC-mass spectrometry (MS), after samples burning. Samples showed different colors and forms, the majority of which is yellow (74.0%) or white (20.0%). Samples free of adulterants represented 76.3% of the total. Mean purity of the analyzed drug was 71.3%. Crack cocaine presented no correlations between macroscopic characteristics and purity. Smoke analysis showed compounds found also in the degradation of diesel and gasoline. Therefore, the drug marketed as crack cocaine in São Paulo has similar characteristics to coca paste. High purity can represent a greater risk of dependency and smoke compounds are possibly worsening drug health impact.