Troubleshooting Odds and Ends to Close Out 2023: Questions from the Mailbag, and Second Opinions

Systematic approach to treatment of enantiomeric separations in capillary electrophoresis and liquid chromatography.: I. Initial evaluation using propranolol and dansylated amino acids

Natural polysaccharides are important active biomolecules. However, the analysis and structural characterization of polysaccharides are challenging tasks that often require multiple techniques and maps to reflect their structural features. This study aimed to propose a new heart-cutting two-dimensional liquid chromatography (2D-LC) method for separating and analyzing polysaccharides to explore the multidimensional information of polysaccharide structure in a single map. That is, the first-dimension liquid chromatography (1D-LC) presents molecular-weight information, and the second-dimension liquid chromatography (2D-LC) shows the fingerprints of polysaccharides. In this 2D-LC system, the size-exclusion chromatography-hydrophilic interaction chromatography (SEC-HILIC) model was established. Coupling with a charged aerosol detector (CAD) eliminated the need for the derivatization of the polysaccharide sample, allowing the whole process to be completed within 80 min. The methods were all validated in terms of precision, linearity, stability, and repeatability. The capability of the new 2D-LC method was demonstrated in determining various species of natural polysaccharides. Our experimental data demonstrated the feasibility of the whole systematic approach, opening the door for further applications in the field of natural polysaccharide analysis.

The rationale behind the measurement of anti-tuberculous (Anti-TB) drugs in biological fluids, especially in the management of tuberculous meningitis (TBM), has been briefly discussed. A short review on methods available for laboratory use has been presented. Finally a systematic approach using liquid chromatography (LC) is presented for consideration. This approach involves a preliminary organic solvent extraction of pyrazinamide (PZA) and rifampicin (RIF) and its 25-desacetyl metabolite, 25-DRIF, from cerebrospinal fluid (CSF) or plasma samples. The drugs and their internal standards are separated on a RPB precolumn linked to a μBondapak cartridge in a Zmodule by a gradient solvent program which delivers 6% to 48% v/v of acetonitrile in a phosphate buffer (10 mM KH2PO4 pH3.5) in 10 min at 1.5 ml/min. The eluate is detected at 215 nm. This LC system can also resolve 2 other anti-TB drugs (isoniazid and p-aminosalicylic acid). Patients with TBM were treated with various combinations of anti-TB dr...

Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics.

Tryptamines represent a group of hallucinogenic new psychoactive substances with increasing prevalence. Unfortunately, only limited data concerning their toxicology and bioanalysis are available as tryptamines are not included in routine screening procedures in many laboratories. In order to expand the current knowledge, we report a non-fatal clinical toxicology case involving the synthetic tryptamine 4-HO-MET (4-hydroxy-N-methyl-N-ethyl-tryptamine, 3-{2-[ethyl(methyl)amino]ethyl}-1H-indol-4-ol, metocin or methylcybin). As only blood of the intoxicated patient was available, our systematic blood plasma screening approaches based on gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) coupled to low-resolution linear ion trap mass spectrometry (ITMSn) or high-resolution tandem mass spectrometry (HRMS-MS) were conducted. The ingestion of the synthetic tryptamine 4-HO-MET could be revealed by blood plasma analysis using both LC-based systematic screening approaches. However, 4-HO-MET was not detected by GC-MS. Furthermore, the detection of metabolites, which may be used to confirm an intake of the parent compound 4-HO-MET, was only successful using LC-HRMS-MS most probably due to its increased sensitivity compared to LC-ITMSn. A total of four metabolites were detected in blood, including N-demethyl-, oxo- and hydroxy-4-HO-MET, as well as the N-oxide. Finally, LC-HRMS-MS analysis revealed a plasma concentration of 193 ng/mL for 4-HO-MET using the standard addition method. The presented data may help clinical and forensic toxicologists with the interpretation of future cases involving synthetic tryptamines, especially if only blood samples are available.

ABSTRACTA systematic approach for identifying and quantifying molecular components of complex organic aerosol mixtures is presented. The approach combines methods developed previously for derivatizing carbonyl, hydroxyl, carboxyl, and ester functional groups, which are commonly present in oxidized organic aerosol, with liquid chromatography, UV detection, and chemical ionization-ion trap mass spectrometry. The original derivatization-spectrophotometric methods were modified for compatibility with liquid chromatography and then evaluated by analyzing a variety of standard compounds that contain one or more functional groups. Detection limits for carbonyl, hydroxyl, carboxyl, and ester analysis are approximately 0.003, 0.02, 0.01, and 1 nmole, respectively. Mass spectral analysis of derivatives using isobutane and ammonia as reagent gases for chemical ionization can be used to determine compound molecular weight, and characteristic fragmentation patterns provide structural information for use in compound id...

Practical limits to column performance in liquid chromatography - Optimal operations.

A new ultra HPLC (UHPLC) method using both MS and fluorescence detection (FD) was developed for the determination of five fluoroquinolones in wastewaters. Systematic method development approach was compared with a conventional one. During the systematic approach, a possibility of automatic switching among four independent analytical columns of different chemistries has been used. Acidic as well as basic pH using ACN and methanol as organic modifiers was tested. The best separation of fluoroquinolones was obtained on phenyl analytical column at pH 10.5, which is a completely novel approach for separation of fluoroquinolones. Further, a new SPE procedure was developed for the sample preparation using basic pH as well. The sensitivity and selectivity of FD and MS detection were compared. FD at basic pH 10.5 demonstrated lower sensitivity than at acidic pH, which is conventionally performed. At basic pH, UHPLC-MS/MS was found about two orders of magnitude more sensitive than FD. Both methods were validated and subsequently UHPLC-FD method was used for the evaluation of stability of fluoroquinolones. UHPLC-MS/MS method was used for the analysis of wastewater samples. Norfloxacin and ciprofloxacin were detected in samples of influent and effluent from wastewater treatment plant. Ofloxacin was detected only in influent from wastewater treatment plant.

Advances in forest tree genomics

Systematic approach to treatment of enantiomeric separations in capillary electrophoresis and liquid chromatography II. A study of the enantiomeric separation of fluoxetine and norfluoxetine

Liquid chromatography (LC) is a technique widely used to identify and quantify organic compounds in a complex mixture. Typical operations of high-performance liquid chromatography (HPLC) involve continuous use of harmful solvents. Replacing these harmful solvents with safer alternatives will provide significant environmental, health, and safety benefits. In this work, a systematic approach for searching safer solvent blends to replace acetonitrile for reversed-phase (RP) HPLC operations is presented. GreenScreen® for Safer Chemicals was used as the first filter to down-select safer solvent candidates from thousands of chemicals based on their safety ratings. A list of LC operation parameters was then employed to determine final solvent candidates. Finally, Hansen Solubility Parameters in Practice (HSPiP) software was utilized to identify the most probable compositions of blends from these solvents for actual LC testing. It was found that a blend of 75% ethanol and 25% methyl acetate by volume provided the chromatograms with the best performance, which had similar response factors and column efficiency compared to acetonitrile when surrogate food additives, dyes, and water pollutants were tested, suggesting that this solvent blend is a potential safer alternative to replace acetonitrile for certain LC applications.

HPLC method development and validation plays an important role in the discovery, manufacturing, development of pharmaceutical products. HPLC methods are able to separate, detect and quantify various drugs their related substances, degraded products and impurities that may be introduced during the synthesis of drug substance. Here in this review various strategies for method development and validation on HPLC are well described. Along with this systematic approach for method validation is also described. Method validation establishes the performance characteristics and limitations of the developed method. Optimization of chromatographic conditions includes fixation of parameters like mobile phase, stationary phase, detection wavelength, elution mode that must affords to system suitability as well as stability of drugs, degradants, impurities. Force degradation studies helpful in the development and validation of stability indicating assay. They also demonstrates the specificity while developing stability indicating assay.

Driving is a complex task, requiring the cooperation of several different cognitive and psychomotor functions. Experimental studies have shown that drugs can have a detrimental influence on capacities that are essential for driving. Epidemiological studies have shown that drugs (not including alcohol) are found in 1–11% of the general driving population, in 6–53% of drivers who are injured or killed in traffic crashes, and in 55–99% of drivers who are suspected of driving under the influence of drugs (DUID). Drivers who are under the influence of drugs have a higher risk of being involved in, or being responsible for, a crash. Responsibility analyses show that driving under the influence of several drugs increases the risk of being responsible for a crash. Three types of DUID legislation exist: “impairment” legislation, “ per se ” legislation, and the “two‐tier” approach. In impairment legislation, the prosecution must demonstrate that the driver was impaired or “under the influence”. A “ per se ” law prohibits driving if drugs are present in blood, serum, plasma, or oral fluid (OF) above a certain threshold concentration. In the two‐tier approach, the per se and impairment approach are combined. In recent years, OF has been used more and more as biological matrix for roadside drug screening. Recent multi‐analyte methods developed for confirmation analysis, either on blood or on OF, use liquid chromatography (tandem) mass spectrometry.

Historical and archaeological textiles: An insight on degradation products of wool and silk yarns

Driving is a very complex task, requiring the cooperation of several different cognitive and psychomotor functions at once. Experimental studies have shown that drugs can have a detrimental influence on capacities that are essential for driving. Cannabis, for example, can acutely reduce some cognitive and psychomotor skills, and these effects are mostly dose dependent. Some deleterious effects of cannabis appear to be additive or even synergistic with those of alcohol, and the combination of both substances results in a prolongation as well as enhancement of their effects. Epidemiological studies have shown that drugs are prevalent in 1–12% of the general driving population, in 6–50% of drivers who are injured or killed in traffic crashes, and in 55–99% of drivers who are suspected of driving under the influence of drugs. The most prevalent drug in most of these studies is cannabis. Drivers who are under the influence of drugs have a higher risk of being involved in or being responsible for a crash. Responsibility analyses show that driving under the influence of cannabis increases the risk of being responsible for an accident, and that this risk increases with increasing cannabis concentration in the blood, indicating a causal relationship between cannabis and crashes. Two major types of driving‐under‐the‐influence of drugs (DUID) legislation exist: “impairment” legislation and “ per se ” legislation. In impairment legislation, the prosecution must demonstrate that the driver was impaired, not fit to drive, or “under the influence”. A “ per se ” law prohibits driving if drugs are present in blood, serum, plasma, or oral fluid above a certain threshold concentration. Since the prosecution does not have to prove that the driver was impaired, this kind of legislation facilitates enforcement. In recent years, the interest in the use of oral fluid as biological matrix for roadside drug screening has increased significantly, as this matrix displays some particularly interesting properties. However the currently available on‐site oral fluid drug testing devices are not reliable enough to be recommended for roadside screening for drivers. Recent methods developed for confirmation analysis, either on blood or on oral fluid, use liquid chromatography (tandem) mass spectrometry (LC‐MS(MS)).