Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests.

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and (2)D retention time opening interesting perspectives for use in QA/QC testing.

Sex-specific seasonal variations in the fatty acid and carotenoid composition of sea cucumber gonads and implications for aquaculture.

Fatty acids and carotenoids are known to have roles in embryonic and larval development of sea cucumbers, but their changes in gonads during gametogenesis have not yet been studied. To improve our knowledge of the reproductive cycle of sea cucumbers in an aquaculture perspective, we collected 6-11 individuals of the species Holothuria (Panningothuria) forskali Delle Chiaje, 1823 approximately every 2 months from December 2019 to July 2021 east of the Glenan Islands (Brittany - France; 47.710°N, 3.948°W) at a depth of 8-12 m. Our results show that soon after spawning, sea cucumbers take advantage of an increased food availability in spring to rapidly and opportunistically accumulate nutrients in the form of lipids in their gonads (from May to July) and then slowly elongate, desaturate and probably rearrange fatty acids within lipid classes for the next reproductive season according to the specific requirements of both sexes. In contrast, acquisition of carotenoids occurs synchronously with gonads filling and/or through the reabsorption of spent tubules (T5), thus revealing little seasonal variations at the scale of the entire gonad in terms of relative abundance in both sexes. All results suggest that gonads are fully replenished with nutrients by October and that broodstock for induced reproduction could be captured at this moment and kept until the production of larvae is required. Maintaining broodstock for consecutive years would probably be a higher level challenge as the dynamics of tubule recruitment are not fully understood and seem to last for several years. Supplementary Information: The online version contains supplementary material available at 10.1007/s00227-023-04198-0.

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

Untargeted flavor profiling of lager beers by stir bar sorptive extraction -capillary gas chromatography - time-of-flight mass spectrometry: High analytical performance with a green touch.

Beer is one of the most popular beverages in the world and its complex flavor is widely appreciated. Beer flavor profiling is important for brewers to optimize beer production and to guarantee odor quality and taste stability of the final products. This is especially the case for pale lager beers that represent the beer type with the largest worldwide production volume. In this study, the combination of stir bar sorptive extraction (SBSE) with capillary gas chromatography (GC) hyphenated to time-of-flight mass spectrometry (TOFMS) was used to perform a detailed aroma profiling of lager beer samples originating from Belgium, The Netherlands, and France. A generic SBSE method was applied resulting in a very broad extraction coverage of odor solutes, while the extraction process is miniaturized, unattended and solventless, meeting green analytical chemistry requirements. Using GC-TOFMS analysis operated in untargeted mode, MS deconvolution and statistical data analysis, with principal component and hierarchical clustering analysis, it was possible to clearly differentiate brands and origins of the beer samples and to identify marker compounds for flavor profiling of these closely related beer samples. An extended database of beer aroma compounds was created. The developed method can be applied in beer quality optimization and quality control in routine laboratories.

Analysis of potential genotoxic impurities in pharmaceuticals by two-dimensional gas chromatography with Deans switching and independent column temperature control using a low-thermal-mass oven module.

The analysis of potential genotoxic impurities (PGIs) in active pharmaceutical ingredients (APIs) is a challenging task. The target limit of detection for a PGI in an API is typically 1 ppm (1 microg/g API). This is about 500 times lower than for classical impurity analysis. Consequently, analytical methods for trace analysis, mostly in combination with MS detection, need to be applied for the qualitative and quantitative determination of these impurities. A two-dimensional capillary GC method is presented that can be used for the determination of some target PGIs. A concentrated solution of the API sample is directly introduced in the GC-MS system, using an apolar column for first-dimension separation. The fraction (heart-cut) containing the PGIs is transferred to a second capillary column, installed in a low-thermal-mass oven (LTM). The LTM focuses the heart-cut(s) and allows independent temperature-programmed analysis with a polar second-dimension column. The API, solvent, and derivatization agents are not introduced in the second column or in the MS detector, avoiding contamination, column degradation, and target analyte peak detection/integration issues. The performance of this set-up is illustrated by the analysis of some Michael-reactive acceptor PGIs and haloalcohols in carbamazepine as test matrix. Excellent reproducibility (<10% RSD) at the low parts per million level and low detection limits (<1 ppm) were obtained.

Public research funding and pharmaceutical prices: do Americans pay twice for drugs?

In the debate over prescription drug pricing, some pharmaceutical industry critics claim that U.S. taxpayers pay twice for costly therapies, because publicly supported research is a major contributor to drug discovery and American taxpayers are inadequately rewarded for their research investment due to high drug prices. In fact, the empirical evidence supporting these claims is weak, and the pay twice argument distracts from important efforts to ensure that impactful new drugs continue to be developed and made widely available to patients who need them.

Fractionated stir bar sorptive extraction using conventional and solvent-assisted approaches for enhanced identification capabilities of aroma compounds in beverages.

For successful profiling of aroma carriers in food samples, a highly efficient extraction method is mandatory. A two-step stir bar sorptive extraction (SBSE) approach, namely fractionated SBSE (Fr-SBSE), was developed to improve both the organoleptic and the chemical identification of aroma compounds in beverages. Fr-SBSE consists of two multi-SBSE procedures (mSBSE) performed sequentially on the same sample. The first extraction consists of a conventional mSBSE using three polydimethylsiloxane (PDMS) stir bars (1stmSBSE). This is followed by a solvent-assisted mSBSE performed on the same sample using three solvent-swollen PDMS stir bars (2nd SA-mSBSE). The 1stmSBSE mainly extracts apolar/medium polar solutes with log Kow values >2, while the 2nd SA-mSBSE mainly extracts polar solutes with log Kow values <2. After this two-step fractionation procedure, either thermal desorption (TD) or liquid desorption - large volume injection (LD-LVI), followed by GC-MS is performed on each set of three stir bars. A real-life sample of roasted green tea was used for method development. The performance of the Fr-SBSE method is further illustrated with sensory evaluations and GC-MS analysis for a stout beer sample. Compared to an extraction procedure with SA-mSBSE only, Fr-SBSE including a 1stmSBSE and a 2nd SA-mSBSE reduced co-elution of aroma compounds in the chromatograms and was capable of providing improved mass spectral quality for identification of 17 additional compounds in roasted green tea, and 12 additional compounds in stout beer, respectively. Moreover, odor description and characterization were clearly improved.

Results of a portfolio approach to intramural research funding at an academic medical center.

In response to stagnant Federal grant funding levels and to catalyze early stage or high-risk research not currently supported by the NIH, many academic medical centers (AMCs) provide supplemental intramural funding to faculty investigators. However, it can be challenging to decide how to deploy these funds for maximum impact. We conducted a retrospective, descriptive analysis to explore trends in applications and awards associated with an institution-wide intramural funding center at a major U.S. AMC. From 2010 to 2017, the Brigham Research Institute at Brigham and Women's Hospital awarded a total of 354 grants totaling over $9 million to affiliated researchers through six distinct and complementary grant programs. The number of applicants remained essentially stable, despite expansion of the funding program portfolio. Distribution of applicants and awardees by academic rank and gender generally reflected that of medical school faculty at large. This descriptive analysis demonstrates interest in a diverse range of intramural funding programs among AMC faculty, and a lack of overt rank or gender bias in the programs' awardees. However, it highlights the institution's need to better understand the amount of residual unmet demand for intramural funding; the degree to which underrepresented constituencies can and should be actively supported; and the "return on investment" of these grants.

Respective contribution of urban wastewater and mangroves on nutrient dynamics in a tropical estuary during the monsoon season.

Estuaries of Southeast Asia are increasingly impacted by land-cover changes and pollution. Here, our research objectives were to (1) determine the origins of nutrient loads along the Can Gio estuary (Vietnam) and (2) identify the processes that affect the nutrient pools during the monsoon. We constructed four 24-h time-series along the salinity gradient measuring nutrient concentrations and stable isotopes values. In the upper estuary, urban effluents from Ho Chi Minh City were the main input of nutrients, leading to dissolved oxygen saturation <20%. In the lower estuary, ammonium and nitrite concentration peaks were explained by mangrove export. No contribution from aquaculture was detected, as it represents <0.01% of the total river discharge. Along the salinity gradient, nutrient inputs were rapidly consumed, potentially by phytoplankton while nitrate dual-stable isotopes indicated that nitrification occurred. Thus, even in a large and productive estuary, urban wastewater can affect nutrient dynamics with potentially important ecological risks.

Study on the migration of bisphenol-A from baby bottles by stir bar sorptive extraction-thermal desorption-capillary GC-MS.

Migration of bisphenol-A (BPA), the principal monomer of polycarbonate (PC) baby bottles, was investigated using an aqueous migration simulant. BPA was determined in 200 mL water samples using stir bar sorptive extraction (SBSE) after in situ derivatization with acetic acid anhydride followed by thermal desorption (TD)-capillary GC-MS. The concentration of BPA was calculated using the deuterated internal standard d6-BPA. Calibration for BPA was shown to be linear in a concentration range from 1 ng/L to 10 microg/L with a correlation coefficient >0.99. The LOD for BPA (as acetate) was 0.12 ng/L and LOQ 0.40 ng/L (ppt). PC bottles were heated in a water bath and in a microwave oven at four different temperatures (37, 53, 65, and 85 degrees C). The higher the temperature, the more the BPA was released, and after a few heating cycles, the released concentrations became constant. At normal use, i.e. at 37 degrees C, concentrations are ca. 10 ng/L. No significant difference was noted between water bath and microwave heating illustrating that migration of BPA is mainly temperature dependent.

The acetonitrile shortage: is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes?

In hydrophilic interaction chromatography (HILIC), best results are obtained with high concentrations of ACN. In the framework of green chromatography and the present shortage and very high price of this hazardous solvent, reversing the stationary phase to apolar and the mobile phase to aqueous can be of interest for several applications. The features of the aqueous RP technique called per aqueous LC (PALC) are illustrated with the analysis of catecholamines, nucleobases, acids, and amino acids. The ca. three-fold higher viscosity of water compared to ACN has consequences on the shape of the Van Deemter plot. For dopamine (N = 26.450 on a 25 cm x 4.6 mm id, 5 microm bare silica column), a reduced plate height of 1.9 at an u(opt) of 0.3 mm/s was calculated. The plate number, however, strongly depends on pH and ionic strength. As in RP separations, retention is shortened by adding an organic modifier. In the framework of green chromatography, the biodegradable ethanol was used. On the other hand, retention increased by lengthening the carbon chain of ion-pair reagents supporting the RP mechanism as well.

A worldwide reliable indicator to differentiate wild vs. farmed Penaeid shrimps based on 207 fatty acid profiles.

Shrimps and prawns are especially subject to food fraud, which has consequences not only on the economy but also represents a potential risk for public health. Fatty acids (FA) of Penaeid shrimps have been largely explored in the literature, and although they are unable to discriminate shrimps geographical origin or species, they might provide an interesting tool to distinguish their production method (wild vs. farmed). The present study is based on a literature compilation of Penaeid shrimp FA profiles encompassing all continents and 28 species. It reveals that the ratio of FA 18:2ω6 + FA 18:3ω3 / FA 16:1ω7 can differentiate wild vs. farmed Penaeid shrimps with 100% accuracy within the 207 FA profiles of the dataset considered. Assuming a normal distribution of the dataset, 94.4% of the farmed shrimps population is expected to exhibit a ratio above 2.92, and 99.7% of the wild shrimps population is expected to fall below 2.92.

Fatty acids, C and N dynamics and stable isotope ratios during experimental degradation of shrimp pond effluents in mangrove water.

Intensive shrimp farming generates high loads of wastewaters that are released along tropical coastlines with potential impacts on the ecosystems. In this study, we used an experimental approach to analyze the behavior of shrimp pond effluents released in the Can Gio mangrove waterways (Southern Vietnam). We incubated shrimp pond effluents (EF), river water (RV), and a mixture of both (MI; 90% RV + 10% EF) in a dark room and measured fatty acid (FA) compositions, C and N concentrations and stable isotopes ratios (δ13C and δ15N) of suspended particulate matter during 16 days. Fatty acid concentrations rapidly decreased in EF with a 50% loss of FA during the first 24 h of the experiment and a 75% loss after 4 days of incubation. Proportions of the FA 18:1ω7 increased in MI during incubation, suggesting that this FA may be used as a tracer of anthropogenic substances release in marine environments.

Features of a micro-gas chromatograph equipped with enrichment device and microchip plasma emission detection (muPED) for air monitoring.

A field portable gas chromatograph (GC) was constructed allowing the enrichment of organic solutes from air samples on a miniaturized chemical trap and the subsequent gas chromatographic analysis on a resistively heated capillary column. The heart of the system is an integrated chip-based plasma emission detector (muPED). As a non-selective detector, the sensitivity is similar to that of a flame ionization detector (FID). The detector shows good selectivity for phosphorus, sulfur and chlorine-containing compounds with relative selectivities of ca. 5 x 10(5) gP gC(-1), 50 gS gC(-1) and 10(2) gCl gC(-1). The lifetime of the plasma chip under air monitoring conditions exceeded 3000 analyses.

Improved accuracy in the determination of polycyclic aromatic hydrocarbons in air using 24 h sampling on a mixed bed followed by thermal desorption capillary gas chromatography-mass spectrometry.

A new sampling method for the determination of polycyclic aromatic hydrocarbons (PAHs) in ambient air is described. The method is based on active sampling on sorption tubes consisting of polydimethylsiloxane (PDMS) foam, PDMS particles and a TENAX TA bed. After sampling, the solutes are quantitatively recovered by thermal desorption and analysed by capillary GC-MS. The new sampling method has been compared to the classical method using high-volume sampling on a glass fiber filter followed by polyurethane foam for 24h sampling of ambient air. Volumes enriched were 144 l on the mixed bed and 1296 m3 with the classical method. The concentrations measured using the new method were significantly higher that the values obtained using the classical method, i.e. a factor 1.2-3 for the high molecular weight PAHs and up to 35 times for naphthalene and 23 times for acenaphthene. The total toxicity equivalence value (TEQ) for PAHs was ca. two times higher compared to the conventional method, illustrating that the concentrations of PAHs in ambient air have been underestimated until now. Some figures of merit (mean value for 17 PAHs) of the method are repeatability 7.4%, detection limit 13 pg/m3, accuracy 105.6% and linearity 0.996. The method also opens interesting perspectives for the determination of other semi-volatile persistent organic pollutants (POPs) in air as illustrated with the analysis of polychlorinated biphenyls (PCBs) at a workplace during removal of transformer oil.

Comparison of one-dimensional and comprehensive two-dimensional separations by gas chromatography.

Comprehensive two-dimensional gas chromatography (GC x GC) can reveal information on the composition of a sample in a way that cannot be done by one-dimensional GC (1D-GC). GC x GC also offers much greater control of chromatographic selectivity based on molecular structure. However, in spite of more than 15 years of claims of the ability of GC x GC to resolve an overwhelmingly larger number of peaks than 1D-GC, and in spite of the theoretically proven potential of GC x GC to have an order of magnitude larger peak capacity than 1D-GC, the peak capacity of currently practiced GC x GC does not generally exceed the peak capacity attainable from 1D-GC with the same analysis time and the same minimal detectable concentration (MDC). The methodology for comparing the peak capacity of GC x GC to 1D-GC is described. The comparison of the performance of GC x GC to 1D-GC shows that the modulator is the key bottleneck limiting the performance of existing GC x GC. To realize the full potential of GC x GC, duration of injection from a modulator into the second-dimension column should be reduced by an order of magnitude or more. Use of powerful data analysis techniques such as peak deconvolution in both dimensions can further increase resolving power of GC x GC.

Creating a fatty acid methyl ester database for lipid profiling in a single drop of human blood using high resolution capillary gas chromatography and mass spectrometry.

Capillary gas chromatography (CGC) in combination with mass spectrometry (MS) was optimized for the separation and detection of the fatty acids occurring in the lipid fraction of blood. A fingertip blood sample (ca. 50 microL) was transesterified into the methyl esters and analyzed on a 100 m x 0.25 mm ID column coated with a biscyanopropyl polysiloxane (HP-88) stationary phase. The method was retention time locked. Programmed temperature vaporization injection (PTV) in the solvent venting mode was applied to minimize the sample size, while maintaining high sensitivity. The total analysis time was ca. 60 min. Retention times and both electron impact (EI) and positive chemical ionization (PCI) mass spectrometry were combined to elucidate the fatty acids according to alkyl chain, degree of unsaturation and position of the double bonds. Using extracted ion chromatograms about 100 fatty acids and related compounds were detected in blood samples and most of them were identified. This work resulted in a very large fatty acid methyl esters database, containing retention time and mass spectral information that will be applied to metabolomic studies.

Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension.

The design of a new interface for comprehensive two-dimensional liquid chromatography (LC x LC) is described. To the conventionally used LC x LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC x 2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC x 2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample.

Chemotaxonomy of bacteria by comprehensive GC and GC-MS in electron impact and chemical ionisation mode.

The analysis of the cellular lipidic fraction of bacteria is described. After hydrolysis and methylation, the fatty acid methyl esters (FAMEs) are determined by 1-D GC using the Sherlock MIDI bacteria identification system, by comprehensive GC (GC x GC) and by GC-MS in electron impact (EI) and positive chemical ionisation (PCI) mode. With GC x GC, the enhanced selectivity and group type separation provides a more complete elucidation of the fatty acids in microorganisms. GC-EI-MS and GC-PCI-MS were helpful for confirmation. The bacteria selected in this study are Brevundimonas diminuta, Chryseobacterium gleum and Stenotrophomonas maltophilia.

Development and validation of an automated static headspace gas chromatography-mass spectrometry (SHS-GC-MS) method for monitoring the formation of ethyl methane sulfonate from ethanol and methane sulfonic acid.

An automated sample preparation and analysis procedure was developed to monitor the formation of ethyl methane sulfonate from reaction mixtures containing ethanol and methane sulfonic acid. The system is based on a liquid handling robot combined with a static headspace module. The formed ethyl methane sulfonate is analysed after derivatisation with pentafluorothiophenol using static headspace-gas chromatography-mass spectrometry (SHS-GC-MS). Using the automated reaction-derivatisation-headspace GC-MS system, the formation of ethyl methane sulfonate can be monitored in different reaction mixtures under different reaction conditions, including temperature, water content and pH. Excellent linearity, repeatability and robustness were obtained, allowing the system to be used in kinetic studies.