Prediction of Vacuum Ultraviolet/Ultraviolet Gas-Phase Absorption Spectra Using Molecular Feature Representations and Machine Learning.

Ultraviolet (UV) absorption spectroscopy is a widely used tool for quantitative and qualitative analyses of chemical compounds. In the gas phase, vacuum UV (VUV) and UV absorption spectra are specific and diagnostic for many small molecules. An accurate prediction of VUV/UV absorption spectra can aid the characterization of new or unknown molecules in areas such as fuels, forensics, and pharmaceutical research. An alternative to quantum chemical spectral prediction is the use of artificial intelligence. Here, different molecular feature representation techniques were used and developed to encode chemical structures for testing three machine learning models to predict gas-phase VUV/UV absorption spectra. Structure data files (.sdf) and VUV/UV absorption spectra for 1397 volatile and semivolatile chemical compounds were used to train and test the models. New molecular features (termed ABOCH) were introduced to better capture pi-bonding, aromaticity, and halogenation. The incorporation of these new features benefited spectral prediction and demonstrated superior performance compared to computationally intensive molecular-based deep learning methods. Of the machine learning methods, the use of a Random Forest regressor returned the best accuracy score with the shortest training time. The developed machine learning prediction model also outperformed spectral predictions based on the time-dependent density functional theory.

Characterization of a Soft Ionization by Chemical Reaction in Transfer Ion Source Hyphenated With Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

A commercially available dielectric barrier discharge ionization (DBDI) source was tested with supercritical fluid chromatography-mass spectrometry (SFC-MS). The compound mixture investigated comprised caffeine, theobromine, theophylline, uracil, testosterone, and pyrene, diluted in methanol. Dynamic response ranges were evaluated with multiple injections at different concentrations. Precision studies demonstrated the robustness and sensitivity of the ionization source across a concentration range of 10-1000 ng/mL. Results from this experiment showed linear regression of 0.99 or greater for all analytes tested over the range with a relative standard deviation (RSD) of less than 10% down to 10 ng/mL for all analytes except theobromine, which had an RSD of less than 10% down to 25 ng/mL. Notably, this study marks the first investigation of sensitivity for coupling a commercial DBDI source with SFC; a limit of detection less than 1 ng/mL was achieved for all compounds. This study demonstrates chromatographic separation by SFC and MS analysis for compounds that ionize poorly using traditional atmospheric pressure ionization, such as polycyclic aromatic hydrocarbons. Combining SFC with the DBDI source opens promising avenues for analyzing compounds that were previously challenging to characterize with standard atmospheric pressure ionization techniques.

Investigation of operational fundamentals for vacuum-assisted headspace high-capacity solid-phase microextraction and gas chromatographic analysis of semivolatile compounds from a model solid sample.

Vacuum-assisted headspace solid-phase microextraction (Vac-HS-SPME) is a technique used to enhance SPME sampling of semi-volatile organic compounds. Here, it was combined with a high-capacity SPME Arrow, which features a larger volume of extraction phase and a more rugged configuration than traditional extraction fibers. An in-depth assessment of the critical parameters was conducted to achieve optimal extraction of representative compounds from a model solid sample matrix (Ottawa sand). Operational fundamentals investigated included the types of seals needed to create a leak-free environment under vacuum conditions; the magnitude of the vacuum applied and time needed to activate the Vac kinetics; order of sample vial preparation methods (VPMs); and other standard variables associated with extract analysis by gas chromatography-mass spectrometry. When exploring the limits of sample VPMs, results indicated an ideal workflow requires the solid sample to be spiked before sealing the vial, allow the sample to rest overnight, then apply vacuum at a pressure of -677 mbar (out of -789 mbar maximum possible vacuum with pump and compressor used), exerted on the vial for 90 s. This work provides the necessary workflow for the optimization of Vac-HS-SPME sampling of analytes from solid matrices.

Unraveling the Complex Composition of Produced Water by Specialized Extraction Methodologies.

Produced water (PW), a waste byproduct of oil and gas extraction, is a complex mixture containing numerous organic solubles and elemental species; these constituents range from polycyclic aromatic hydrocarbons to naturally occurring radioactive materials. Identification of these compounds is critical in developing reuse and disposal protocols to minimize environmental contamination and health risks. In this study, versatile extraction methodologies were investigated for the untargeted analysis of PW. Thin-film solid-phase microextraction with hydrophilic-lipophilic balance particles was utilized for the extraction of organic solubles from eight PW samples from the Permian Basin and Eagle Ford formation in Texas. Gas chromatography-mass spectrometry analysis found a total of 266 different organic constituents including 1,4-dioxane, atrazine, pyridine, and PAHs. The elemental composition of PW was evaluated using dispersive solid-phase extraction followed by inductively coupled plasma-mass spectrometry, utilizing a new coordinating sorbent, poly(pyrrole-1-carboxylic acid). This confirmed the presence of 29 elements including rare earth elements, as well as hazardous metals such as Cr, Cd, Pb, and U. Utilizing chemometric analysis, both approaches facilitated the discrimination of each PW sample based on their geochemical origin with a prediction accuracy above 90% using partial least-squares-discriminant analysis, paving the way for PW origin tracing in the environment.

Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions.

In the host, pathogenic microorganisms have developed stress responses to cope with constantly changing environments. Stress responses are directly related to changes in several metabolomic pathways, which could hamper microorganisms' unequivocal identification. We evaluated the effect of various in vitro stress conditions (acidic, basic, oxidative, ethanolic, and saline conditions) on the metabolism of Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa, which are common lung pathogens. The metabolite profiles of the bacteria were analyzed using liquid chromatography coupled to triple quadrupole and quadrupole time-of-flight mass spectrometry. The advantages of targeted and untargeted analysis combined with univariate and multivariate statistical analysis (principal component analysis, hierarchical cluster analysis, partial least square discriminant analysis, random forest) were combined to unequivocally identify bacterial species. In normal in vitro conditions, the targeted methodology, based on the analysis of primary metabolites, enabled the rapid and efficient discrimination of the three bacteria. In changing in vitro conditions and specifically in presence of the various stressors, the untargeted methodology proved to be more valuable for the global and accurate differentiation of the three bacteria, also considering the type of stress environment within each species. In addition, species-specific metabolites (i.e., fatty acids, polysaccharides, peptides, and nucleotide bases derivatives) were putatively identified. Good intra-day repeatability and inter-day repeatability (< 10% RSD and < 15% RSD, respectively) were obtained for the targeted and the untargeted methods. This untargeted approach highlights its importance in unusual (and less known) bacterial growth environments, being a powerful tool for infectious disease diagnosis, where the accurate classification of microorganisms is sought.

A review of ASTM D7979 for the analysis of per- and polyfluorinated alkyl substances in non-potable water by co-solvation with methanol and using liquid chromatography-tandem mass spectrometry.

Per- and polyfluorinated alkyl substances are large class of man-made compounds known in the media as "forever chemicals". In 2015, ASTM International published ASTM D7979, for the analysis of per- and polyfluorinated alkyl substances in non-potable water samples. This method extracts the substances by co-solvation with methanol and measures targeted compounds using liquid chromatography-tandem mass spectrometry. ASTM D7979 is a performance-based method that analyzes 31 compounds plus 14 isotopically labeled surrogates. The minimum reporting limit is approximately 10 ng/L with an analytical range of 10-200 ng/L for most compounds. Expected recovery of surrogates and spiked matrices is 70-130%. Samples containing high suspended solids can be analyzed with minimal interferences and potential loss of analyte. The method is consistent with ASTM and EPA's sustainable development goals by using reduced volumes of sample, solvent, and minimizing hazardous solvents and sample preparation materials while maintaining data quality and detection limits that are suitable for the intended use. This paper covers the rationale, outlines some of the challenges associated with analysis of per- and polyfluorinated alkyl substances, and describes the steps taken by the ASTM Committee D19 task group to develop, optimize, and validate this method.

Target profiling of beer styles by their iso-α-acid and phenolic content using liquid chromatography-quadrupole time-of-flight-mass spectrometry.

Beer styles show wide variation in color, flavor, and clarity, due to differences in their chemical content. Some of the major flavor compounds in beer are isomerized alpha acids and phenolic compounds. These were investigated as potentially discerning features between beer styles. A selection of 32 American beers covering five styles was analyzed using liquid chromatography quadrupole time-of-flight mass spectrometry, which resulted in high mass accuracy chromatograms of the studied analytes. Distinctions between the presence or relative concentrations of certain compounds were observed and related back to brewing ingredients and procedures. For example, vanillin was only observed in stout beers due to the use of roasted barley malts for brewing, while chlorogenic acid isomers were found in two sours at relatively high concentrations (189 and 34 mg/L) because of the fruits used to flavor the beers. Distinctions were further confirmed using multivariate analysis techniques, which separated three of the five beer styles (India pale ales, stouts, and sours).

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis.

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein-based pharmaceuticals. The introduction and maturation of "soft" ionization methods, such as electrospray ionization and matrix-assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis. This review summarizes these challenges and provides an overview of the state-of-the-art in sample preparation and fractionation of proteins for mass spectrometric analysis, with an emphasis on those used for top-down proteomic approaches. Biological fluids, particularly important for clinical and pharmaceutical applications and their characteristics are also discussed. While immunoaffinity-based methods are addressed, more attention is given to non-immunoaffinity-based methods, such as precipitation, coacervation, size exclusion, dialysis, solid-phase extraction, and electrophoresis. These techniques are presented in the context of a significant number of studies where they have been developed and utilized.

Optimization of thin film solid phase microextraction and data deconvolution methods for accurate characterization of organic compounds in produced water.

The continued rise in the extraction of unconventional oil and gas across the globe poses many questions about how to manage these relatively new waste-streams. Produced water, the primary waste by-product, contains a diverse number of anthropogenic additives together with the numerous hydrocarbons extracted from the well. Due to potential environmental hazards, it is critical to characterize the chemical composition of this type of waste before proper disposal or remediation/reuse. In this work, a thin film solid phase microextraction approach was developed and optimized to characterize produced water. The thin film device consisted of hydrophilic-lipophilic balance particles embedded in polydimethylsiloxane and immobilized on a carbon mesh surface. These devices were chosen to provide broad extraction coverage and high reusability. Various parameters were evaluated to ensure reproducible results while minimizing analyte loss. This optimized protocol, consisting of a 15 min extraction followed by a short (3 s) rinsing step, enabled the reproducible analysis of produced water without any sample pretreatment. Extraction efficiency was suitable for both produced water additives and hydrocarbons. The developed approach was able to tentatively identify a total of 201 compounds from produced water samples, by using one-dimensional gas chromatography hyphenated to mass spectrometry and data deconvolution.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography-triple quadrupole mass spectrometry method for determination of intact growth factor proteins.

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.

Evaluation of Different Stationary Phases in the Separation of Inter-Cross-Linked Peptides.

Chemical cross-linking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for cross-linking/MS is the determination of informative low-abundant inter-cross-linked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reversed-phase (RP) separation of inter-cross-linked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) cross-linked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the three columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-cross-linked peptides. Among these three columns, the fluorophenyl column provides additional ion-exchange interaction and exhibits unique retention in separating the cross-linked peptides. The fractioned data was analyzed in pLink, showing the fluorophenyl column consistently obtained more inter-cross-linked peptide identifications than both C18 and biphenyl columns. For the BSA cross-linked sample, the identified inter-cross-linked peptide numbers of the fluorophenyl to C18 column are 136 to 102 in "low confident" results and 11 to 6 in "high confident" results. The fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-cross-linked peptides.

Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on-off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

Characterization of ethoxylated alcohols in friction reducers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) provides detailed information for the analysis of ethoxylated alcohols and polymers. In this study, five friction reducers used in commercial hydraulic fracturing processes were analyzed in their as-received form to identify their ethoxylated alcohol content. The friction reducers were then subjected to lab-simulated downhole conditions. Characterization of friction reducers before and after being subjected to reactive conditions can provide fingerprints associated with produced oilfield waste for source apportionment and information on the stability of these key hydraulic fracturing additives. METHODS: Five different industrially used friction reducers were analyzed for their ethoxylated alcohol content using MALDI-TOF-MS. Three different matrices were assessed for optimal response: α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid with 2,2,2-trifluoroethanol (2,5-DHB + E). Reaction times, temperatures, and sample matrices (deionized water, produced water inorganic, produced water, and produced water + shale core) were varied to assess changes in molecular weight distribution and polydispersity of the ethoxylated alcohols relative to their as-received content. RESULTS: A preference for the 2,5-DHB + E matrix was observed. The friction reducers were found to contain ethoxylated alcohols with carbon chain lengths of 12 and 14 with degrees of ethoxylation ranging from 6 to 18. Upon being subjected to 100°C for 24 hours, the ethoxylated alcohols tended to polymerize further, returning higher average molecular weights. Less polymerization was seen in more complex matrices, as supported by dispersity calculations. CONCLUSIONS: Ethoxylated alcohol content was effectively determined in friction reducers using MALDI-TOF-MS. Although this is not a new technique to characterize ethoxylated alcohols, it has proven to be a quick and effective way to determine ethoxylated alcohol content in friction reducers in complex oilfield matrices. This technique can be used as a rapid and straightforward way to determine ethoxylated alcohol content in friction reducers and hydraulic fracturing wastewater for fingerprinting.

Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

RATIONALE: Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. METHODS: Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined. MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations. RESULTS: For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3- and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed. The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. CONCLUSIONS: The feasibility for top-down quantitative analysis of high-molecular-weight proteins with a triple quadrupole mass spectrometer was demonstrated. Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.

Comparative study of ink photoinitiators in food packages using gas chromatography with vacuum ultraviolet detection and gas chromatography with mass spectrometry.

This work focused on the development and validation of the analytical procedure using gas chromatography equipped with vacuum-ultraviolet detection for the specific and sensitive determination of nine photoinitiators in food packages. Subsequently, a comparison of the combination of vacuum ultraviolet spectroscopy with gas chromatography and a developed gas chromatography with mass spectrometry method was performed. The vacuum-ultraviolet spectra of all tested photoinitiators were collected and found to be highly distinct, even for isomers. Under the optimal conditions, the limits of detection for nine photoinitiators ranged from 1 to 5 mg/L using vacuum ultraviolet detection and from 0.15 to 0.5 mg/L using mass spectrometric detection. Both techniques were successfully applied for screening of photoinitiators in seven kinds of food packages and the obtained data showed good agreement (the relative difference was between 3 and 18%). The variability in concentrations found in triplicate samples was assessed to be below 18%. Predominantly benzophenone was found in all analysed samples in the range of 0.31-4.23 mg/kg. It appears to be preferably selected by food packaging manufacturers. This study proposes a new simple and sensitive technique used for analysis of photoinitiators that could be a good alternative to gas chromatography with mass spectrometry.

Effects of solvent parameters on the electrospray ionization tandem mass spectrometry response of glucose.

RATIONALE: The importance of saccharides, the most abundant biomolecules on Earth, extends beyond their biological roles and to consumer products and industrial processes. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an attractive tool for the analysis of underivatized saccharides (US), but they tend to have relatively low sensitivities due to their low surface activities and lack of easily protonable or deprotonable chemical groups. An understanding of the influences that solvent parameters have on their signal intensities would enhance the usefulness of ESI-MS/MS for their analysis. METHODS: Solutions of glucose, a model analyte for US, in various combinations of solvent, additive, additive concentration, and pH were analyzed by flow injection analysis ESI-MS/MS in both the positive and negative ionization mode. The blank-corrected signal intensities of the solvent parameter combinations were then compared. RESULTS: The addition of acetonitrile led to severe ionization suppression in the positive ionization mode through its competition with glucose for cation adduction. High signal intensity was achieved under wide pH and concentration ranges for methanol: water solutions containing ammonium trifluoroacetate in the positive ionization mode. The highest signal intensities for acetonitrile: water solutions were those containing ammonium formate or lithium fluoride in the negative ionization mode. CONCLUSIONS: An understanding of the influence of solvent parameters on the signal intensity of a given analyte is useful for guiding the selection process of mobile phases/flow solvents that lead to low limits of detection or the minimization of matrix effects by allowing its detection at high dilution factors.

Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry.

Cannabinoids are the primary bioactive constituents of Cannabis sativa and Cannabis indica plants. In this work, gas chromatography in conjunction with triple quadrupole mass spectrometry in multiple reaction monitoring mode was explored for determination of cannabinoids from a surrogate hops matrix. Gas chromatography with mass spectrometry is a reasonable choice for the analysis of these compounds; however, such methods are susceptible to false positives for Δ9-tetrahydrocannabinol, due to decarboxylation of Δ9-tetrahydrocannabinolic acid, its acid precursor, in the hot injection port. To avoid this transformation, the carboxyl group of Δ9-tetrahydrocannabinolic acid was protected through a silylation reaction. Multiple reaction monitoring transitions for both unmodified and silylated cannabinoids were developed and the fragmentation pathways for the different species were assigned. Precision and accuracy were evaluated for cannabinoids spiked into hops at different levels. The developed methods provided good linearity (R2  > 0.99) for all the cannabinoids with a linear range from 0.15 to 20 mg/L, and with limits of detection in the orders of low- to mid-picogram on column. The recoveries for the cannabinoids were generally between 75 and 120%. Precisions (<6% coefficient of variation) were within acceptable ranges.

Complex mixture quantification without calibration using gas chromatography and a comprehensive carbon reactor in conjunction with flame ionization detection.

Quantification of complex carbon-containing mixtures is typically a very time-intensive task with regards to the calibration process. A gas chromatograph with a flame ionization detector yields strong responses to organic compounds and provides a wide linear range over many orders of magnitude; however, responses for highly functionalized and heteroatom-containing compounds can be variable. Here, a commercial Polyarc microreactor unit, placed before the flame ionization detector, was investigated as a means of normalizing carbon response across all compounds. The device includes two catalytic reaction chambers, ultimately converting all carbon atoms to methane evenly for flame ionization detection. Three groups of different complex mixtures from n-alkane to terpene and polymer mixtures were analyzed to evaluate the potential for calibration-free quantitation of the new detector arrangement. We have obtained accurate quantification results without time-consuming calibration processes. The quantification of a terpene mixture and a polymer mixture confirms the ability of the detector for analyzing samples that either have complex physical or structural properties or wide concentration range. In summary, compared to other detectors, this methanizer - flame ionization detection system provides a simplified workflow, which can eliminate calibration steps and increase throughput.

A review of methods for the chemical characterization of cannabis natural products.

Cannabis has garnered a great deal of new attention in the past couple of years in the United States due to the increasing instances of its legalization for recreational use and indications for medicinal benefit. Despite a growing number of laboratories focused on cannabis analysis, the separation science literature pertaining to the determination of cannabis natural products is still in its infancy despite the plant having been utilized by humans for nearly 30 000 years and it being now the most widely used drug worldwide. This is largely attributable to the restrictions associated with cannabis as it is characterized as a schedule 1 drug in the United States. Presented here are reviewed analytical methods for the determination of cannabinoids (primarily) and terpenes (secondarily), the primary natural products of interest in cannabis plants. Focus is placed foremost on analyses from plant extracts and the various instrumentation and techniques that are used, but some coverage is also given to analysis of cannabinoid metabolites found in biological fluids. The goal of this work is to provide a collection of relevant separation science information, upon which the field of cannabis analysis can continue to grow.

An integrated multipath liquid chromatography-mass spectrometry system for the simultaneous preparation, separation, and detection of proteins and small molecules.

A multipath liquid chromatography with mass spectrometry instrument was constructed with the help of restricted access media to online segregate small and large molecules. This liquid chromatography system was custom built with five pumps and three two-position six-port valves to control the flow in a multipath system for the simultaneous analysis of small molecules and proteins. On separate chromatographic channels, small molecules trapped and proteins excluded from the online restricted access media were analyzed downstream using high-efficiency columns and a triple quadrupole mass spectrometer. A model sample, which included five proteins and 22 small molecules with different physicochemical properties, was used to evaluate the system. Following injection, the complete multipath separation and detection was performed in 22 min. Protein exclusion by the restricted access media was not quantitative. Four commercial trap columns were evaluated for their exclusion efficiency toward the proteins. Exclusion efficiency varied from <50% to only a maximum of 75% exclusion across the trap columns tested. An attempt was made to optimize the exclusion efficiency using different flow rates, flow rate gradients, and different additives both in the sample and the mobile phases. Protein exclusion was still erratic and generally nonquantitative.