Single Quadrupole Mass Spectrometry Research Articles

A practical HPLC-MS method for the analysis of nitrosamine drug substance related impurities using an inexpensive single quadrupole mass spectrometer

Nitrosamine drug substance related impurities (NDSRIs) are often analyzed using high performance liquid chromatography (HPLC) with mass spectrometry (MS) detection. Due to high sensitivity requirements, high resolution MS or MS/MS is commonly used. However, it is difficult to implement this type of method for routine analysis at a supply site. Herein, we report a systematic approach to develop and validate a practical, robust, and user-friendly method for the analysis of NDSRIs using an inexpensive single quadrupole MS instrument such as QDa. We used 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro- [1,2,4] triazolo [4,3-a] pyrazine (NTTP) as an example to demonstrate the method development process. By optimizing the HPLC and MS parameters, we were able to develop a simple HPLC-MS method that provides the desired specificity and sensitivity for the analysis of NTTP and can be easily implemented in an analytical lab. The limit of quantitation is 0.5 ng/mL, corresponding to 0.1 ppm with respect to 5 mg/mL sitagliptin. The method has been successfully validated per ICH guidelines.

Comparison of head space solid phase micro extraction with conventional and comprehensive gas chromatography mass spectrometry for volatile profiling of Irish whiskey

Whiskey is a traditional distilled product produced on the island of Ireland that is currently experiencing a major expansion in export volume. Different styles of Irish whiskey exist, however only some very limited published information exists on the volatile congener profile of the different styles of Irish whiskey. Such information is potentially indispensable from a quality, flavour and authenticity perspective. As gas chromatography is the established analytical method of choice to identify volatile congeners, this study compared headspace-solid phase microextraction (HS-SPME) using conventional gas chromatography single quadrupole mass spectrometry, to HS-SPME and HS-SPME-Arrow using comprehensive two-dimensional gas chromatography (GC×GC) modulated using reverse flow in tandem with time-of-flight mass spectrometry (TOFMS) detection. Six representative Irish whiskey samples were evaluated consisting of new make spirit and mature whiskies representing Single Malts, Pot Stills and Blends. The number of volatile congeners identified in these samples by HS-SPME/HS-SPME Arrow GC×GC-TOFMS was approximately twice that detected by conventional HS-SPME GCMS. In total 145 unique individual volatile congeners, excluding ethanol were identified, with the majority consisting of esters, but also benzenes, alcohols, aldehydes, terpenoids, furans, ketones, alkanes, alkenes, norisoprenoids, acetals, acids, lactones and phenols. The use of HS-SPME Arrow GC×GC-TOFMS significantly enhances the number of volatile congeners that can be identified and therefore provides much more information that can convey insights into product quality, consistency, flavour and also for authentication purposes.

Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of 13C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For pre-column derivatization, plasma supernatants were buffered with sodium borate (pH 9.5 −10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole mass spectrometry. We hypothesized that pre-column Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective 13C internal standards, with limits of quantitation beyond physiological ranges. All the 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly that upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. While robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-UV and HPLC-FLD), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

Selectivity of Explosives Analysis with Ambient Ionization Single Quadrupole Mass Spectrometry: Implications for Trace Detection.

Ambient ionization (AI) is a rapidly growing field in mass spectrometry (MS). It allows for the direct analysis of samples without any sample preparation, making it a promising technique for the detection of explosives. Previous studies have shown that AI can be used to detect a variety of explosives, but the exact gas-phase reactions that occur during ionization are not fully understood. This is further complicated by differences in mass spectrometers and individual experimental set ups between researchers. This study investigated the gas-phase ion reactions of five different explosives using a variety of AI techniques coupled to a Waters QDa mass spectrometer to identify selective ions for explosive detection and identification based on the applied ambient ionization technique. The results showed that the choice of the ion source can have a significant impact on the number of ions observed. This can affect the sensitivity and selectivity of the data produced. The findings of this study provide new insights into the gas-phase ion reactions of explosives and could lead to the development of more sensitive and selective AI-based methods for their detection.

Prediction of the Impact of Land Use and Soil Type on Concentrations of Heavy Metals and Phthalates in Soil Based on Model Simulation.

The main objective of this study is to determine the possibility of predicting the impact of land use and soil type on concentrations of heavy metals (HMs) and phthalates (PAEs) in soil based on an artificial neural network model (ANN). Qualitative analysis of HMs was performed with inductively coupled plasma-optical emission spectrometry (ICP/OES) and Direct Mercury Analyzer. Determination of PAEs was performed with gas chromatography (GC) coupled with a single quadrupole mass spectrometry (MS). An ANN, based on the Broyden-Fletcher-Goldfarb-Shanno (BFGS) iterative algorithm, for the prediction of HM and PAE concentrations, based on land use and soil type parameters, showed good prediction capabilities (the coefficient of determination (r2) values during the training cycle for HM concentration variables were 0.895, 0.927, 0.885, 0.813, 0.883, 0.917, 0.931, and 0.883, respectively, and for PAEs, the concentration variables were 0.950, 0.974, 0.958, 0.974, and 0.943, respectively). The results of this study indicate that HM and PAE concentrations, based on land use and soil type, can be predicted using ANN.

Liquid Chromatography Electrospray Ionization Mass Spectrometry Analysis of Camphor and Menthol

Helium is an inactive gas that has been used as a carrier gas in gas chromatography (GC). Due to a recent helium shortage of last several years, a method transfer from GC to high-performance liquid chromatography (HPLC) has been increasingly considered. Here, camphor and menthol, which are used in many pharmaceutical products and have been widely analyzed by GC, were determined by reversed-phase liquid chromatography (LC) coupled with electrospray ionization single quadrupole mass spectrometry (ESI/MS). An excellent linearity was confirmed in the concentration range of 0.5–500 µg/mL for camphor and 1–1000 µg/mL for menthol. Diluted, commercially available eye drops were analyzed with the developed LC-MS method, where both of these compounds were successfully quantitated with a good precision as well as excellent accuracy. The developed LC-MS method can be applied as a promising alternative to the analysis of similar classes of volatile compounds in GC.

Modern methods for the determination of pesticide residues in beekeeping products and for the diagnostics of bee poisoning

Intensification of agricultural production is associated with the use of a significant amount of pesticides, which negatively affects the environment and human health, and food products, including beekeeping products, accordingly require mandatory control of residual amounts of pesticides. This article provides a comparative analysis of the available chromatographic methods for pesticide residue research. The necessity of using modern chromatographic methods to determine residual amounts of pesticides in samples of dead bees and beekeeping products is well-founded. Chromatographic methods of studying these indicators in different types of matrices are a priority. They are effective methods of analysis, widely used due to their versatility - they allow the analysis of complex inorganic and organic compounds in various aggregate states. But one of the most common modern methods for pesticide determination is gas and liquid three-quadrupole tandem chromatography-mass spectrometry (GC and/or LC-MS/MS). The GC-MS/MS method provides quantitative determination of analytes at a level that is an order of magnitude higher than, for example, the gas single quadrupole mass spectrometry method. Modern methods of gas and liquid chromatography in combination with quadrupoletime-of-flight mass spectrometric detection (LC/Q-TOF/MS or GC/ Q-TOF/MS) also allow qualitative and quantitative multicomponent analysis of pesticides in beekeeping products. GC and LC systems combined with high-resolution Orbitrap MS (GC-HRMS(Q-Orbitrap)/LC-HRMS (Q-Orbitrap)) have higher sensitivity, enabling ultra-trace detection, and are the most sensitive screening method for multicomponent determination of pesticide residues. Thus, the latest chromatographic methods are able to meet the needs of analytical testing and research laboratories in the field of food safety, including beekeeping products. Key words: chromatographic methods, thin-layer chromatography, gas chromatography, liquid chromatography, mass spectrometry, multi-component analysis, pesticides, honey, dead bees.

Optimization of a GC-MS method for the detection of bioactive compounds from the green peel of pomelo cultivar ‘PO52’

Pomelo is the largest and most popular citrus fruit known to possess diverse health benefits. Sabah’s fruit basket, Tenom, is famous for pomelo production. Among the pomelo cultivars being planted in Tenom, the PO52 cultivar is the most abundant one. To date, the bioactive compounds of the PO52 cultivar are not well studied. Although numerous aroma-active compounds from the peels of Malaysian pomelo have been reported, there are still many potential non-aromatic bioactive compounds that are yet to be discovered. In this study, we analysed the peel extract of pomelo PO52 using gas chromatography coupled to a single quadrupole mass spectrometry (GC-MS) approach. We first analysed the pomelo peel extracts using two different GC-MS methods, one can detect 45 organic compounds with good separation, while the other can detect phytosterols and pentacyclic triterpenoids in Clinacanthus nutans roots. In our study, these two methods were able to detect bioactive compounds from the pomelo peels but with a longer running time and the generated total ion chromatograms (TIC) were also not smooth. Therefore, we further optimized the two methods by changing the temperature ramping and holding times of the GC-MS. Compared to the first two methods, the optimized method had the shortest total running time (38 min) and the total ion chromatogram (TIC) produced was smooth throughout the run time. Furthermore, bioactive compounds that have not been reported for pomelo peel before, such as osthole, α-tocopherol, gamma-sitosterol, friedelan-3-one and others were also detected using this optimized method. In conclusion, the optimized method is suitable for the detection of non-aromatic compounds present in the PO52 peels that can be subsequently applied in the next stage for large-scale analysis.

A rapid UHPLC-QDa method for quantification of human salivary amino acid profiles

Abnormal salivary amino acid (AA) levels may indicate dysfunction of the body. Being noninvasive, sampling easily and cost-effective of saliva, a rapid, precise and simple analysis method has become very important for quantitative salivary AA profiles. After one-step to precipitate protein, the resultant extraction was derived with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) within 10 min. Quantitation of AA profile was achieved within 6 min in a single run by ultra-high performance liquid chromatography coupled with a single quadrupole mass spectrometry (UHPLC-QDA detector). The method was validated with acceptable accuracy ranging from 80.33 % to 122.31 %, appropriate linearity with the coefficient (R2) more than 0.991, good intra- and inter-day precision, repeatability and stability (RSD < 15 %). The recoveries at three different spiked concentrations ranged over 79.18 %–125.36 % while the matrix effect was from −19.86 % to 11.95 %. This simple, rapid and robust method was successfully applied to quantify human salivary 30 amino acids, in which the levels of taurine, γ-aminobutyric acid, methionine and tryptophan in healthy people were close to the LOQs. Besides, the levels of histidine and cystine were not able to be measured due to their relatively high LOQs, which were considered as the limitations of this developed method.

Direct quantification of PAHs and nitro-PAHs in atmospheric PM by thermal desorption gas chromatography with electron ionization mass spectroscopic detection

In this work, we developed and optimized a method for the analysis of PAHs and nitro-PAHs in atmospheric particulate matter (PM) samples by using thermal desorption gas chromatography coupled with electron ionization single quadrupole mass spectrometry (TD-GC-(EI)-MS). The method uses thermal desorption from a PM on a filter sample as means of sample introduction to a column and obviates the need for complex extraction procedures, which are time-consuming and require environmentally unfriendly solvents. Moreover, the possibility of systematic errors is minimized and a significantly smaller amount of sample is required compared to traditional techniques requiring a pre-extraction step (approx. 10-times). Thirteen PAHs and three nitro-PAHs were used during method development. Although Tenax cartridges are typically used to capture volatile pollutants from the air, we found that glass-wool liner is the most suitable trap for the examined analytes after desorption from a quartz filter. Among the various instrument parameters which were tested and optimized, TD desorption flow and hold time, and temperature of the cooled injection system (CIS) proved to be most critical. We also found out that the matrix effect is especially pronounced in the case of high PM loadings, which should be kept in mind when planning the analysis. After the optimization, standard reference materials (ERM-CZ100 and NIST 1648a) were used for partial method validation and finally, real PM10 and PM2.5 samples from two Slovenian cities were successfully analyzed.

Role of gas chromatography–single quadrupole mass spectrometry in the identification of compounds migrating from polypropylene-based food contact plastics

The migration of volatile and semi-volatile substances from commercially available polypropylene-based food contact materials was studied. Both intentionally and non-intentionally added substances – among them degradation products and contaminants of common additives – were identified. The efficiency and the reliability of tentative identification with GC-EI-QMS measurements followed by NIST mass spectral library searches were also evaluated. In addition to the 24 migrated n-alkanes, 46 compounds were tentatively identified with GC-EI-QMS. Of these, 31 were confirmed with both GC-EI-TOFMS and analytical reference materials. In four cases, however, the tentative identification proved to be wrong. Two compounds were identified despite not being included in the library. Our results prove that GC-EI-QMS and the NIST mass spectral library combined are powerful in the identification of compounds migrating from food contact plastics. But these same results also prove that both reliability and productivity can be improved with GC-EI-TOFMS measurements and consideration of linear retention indices.

Analytical Method Development for 19 Alkyl Halides as Potential Genotoxic Impurities by Analytical Quality by Design

Major issues in the pharmaceutical industry involve efficient risk management and control strategies of potential genotoxic impurities (PGIs). As a result, the development of an appropriate method to control these impurities is required. An optimally sensitive and simultaneous analytical method using gas chromatography with a mass spectrometry detector (GC–MS) was developed for 19 alkyl halides determined to be PGIs. These 19 alkyl halides were selected from 144 alkyl halides through an in silico study utilizing quantitative structure–activity relationship (Q-SAR) approaches via expert knowledge rule-based software and statistical-based software. The analytical quality by design (QbD) approach was adopted for the development of a sensitive and robust analytical method for PGIs. A limited number of literature studies have reviewed the analytical QbD approach in the PGI method development using GC–MS as the analytical instrument. A GC equipped with a single quadrupole mass spectrometry detector (MSD) and VF-624 ms capillary column was used. The developed method was validated in terms of specificity, the limit of detection, quantitation, linearity, accuracy, and precision, according to the ICH Q2 guideline.

Ultrahigh-Performance Supercritical Fluid Chromatography and Detection of Multiple Biogenic Amines in Gentamicin Sulfate: Method Development Using Computer-Assisted Modeling.

In order to solve the problem of difficult separation of various biogenic amines (BAs), which have similar structures or very different polarities, in gentamicin, by conventional liquid chromatography, a new ultrahigh-performance supercritical fluid chromatography (UHPSFC) method was developed. In this method, 10 BAs were derivatized precolumn using dansyl chloride and separated using a UHPSFC system. By computational simulation, complete separation of 10 BAs was successfully achieved. Detection was performed using a photodiode array (PDA) and single-quadrupole mass spectrometry (MS) together with electrospray ionization (ESI). A wide linear range (10-2500 ng/mL) was achieved, with the limits of detection (LODs) between 1.2 and 10.0 ng/mL and the limits of quantification (LOQs) between 5.0 and 25.0 ng/mL. Apart from high sensitivity, this UHPSFC-PDA/ESI-MS detection method also displayed high accuracy, the matrix effect was reduced by an appreciable extent, and the recovery rates of the 10 BAs were between 84.1 and 117.1%. For comparison, high-performance liquid chromatography-tandem mass spectrometry (MS/MS) was also used for the detection of underivatized BAs in gentamicin, showing good linearity and high sensitivity (LODs from 0.05 to 1.00 ng/mL and LOQs from 1.00 to 12.50 ng/mL) for all BAs except for spermine and spermidine. Although single-quadrupole MS is inferior to MS/MS in terms of sensitivity, the UHPSFC method could detect more BAs. It also achieved the quantification limits required for impurity determination, demonstrating a potential strategy to offer a map overview of possible BA presence in fermentation antibiotics.

Safety assessment of citrus and olive by-products using a sustainable methodology based on natural deep eutectic solvents

In this work, the application of betaine-based hydrophilic natural deep eutectic solvents (NADESs) as green extraction solvents was proposed for the first time for the evaluation of twelve pesticides in citrus and olive by-products intended to be applied as potential sources of compounds with neuroprotective activity against Alzheimer Disease. Ultrasound-assisted extraction of selected pesticides was followed by separation and determination using gas chromatography coupled to single quadrupole mass spectrometry. Eight NADESs were tested using different hydrogen bond donors (i.e. citric and lactic acid, fructose, glucose, glycerol, propylene glycol, propionic and butanoic acid). Other factors affecting extraction efficiency were also evaluated using a step-by-step approach. Eight mL of a mixture composed of 60% betaine:propylene glycol NADES at a molar ratio 1:4 and 40% of water, as well as 30 min of ultrasound-assisted extraction were selected as the most adequate conditions. The methodology was validated prior to its application in citrus and olive by-products. Recovery values were between 73 and 115% (RSD% < 20%), while limits of quantification of the method were in the range 8.5–128.8 µg/kg, which demonstrates the suitability of the procedure to determine the selected group of pesticides, usually applied in citrus and olive crops, at the legislated levels. The greenness of the procedure was also evaluated using AGREE calculator. Finally, the whole method was applied for the safety assessment of seven olive leaf samples and seven citrus by-products produced in Spain, finding the presence of several of the evaluated compounds at concentrations higher than the established limits for similar products.

Comparison of gas- and liquid chromatography-mass spectrometry for trace analysis of anilines in groundwater

ABSTRACT Three chromatographic procedures were investigated regarding their potential for the quantification of aniline and 19 of its methylated and chlorinated derivatives in groundwater. These methods were based on liquid-liquid-extraction in combination with gas chromatography and single quadrupole mass spectrometry (GC/MS) according to German standard DIN 38407–16:1999 and its extension using tandem mass spectrometry (GC/MS-MS), both following liquid–liquid extraction, and as third alternative the direct injection of the water sample into a liquid chromatograph coupled to tandem mass spectrometry (LC/MS-MS). Results were compared using fortified water and real-world contaminated groundwater used in an interlaboratory comparison. It could be shown that GC/MS and GC/MS-MS yielded results deviating less than 10% from each other, while all three procedures displayed quantification results deviating less than 15% from the intercomparison reference values in case of each analyte in the concentration range between 1 and 45 µg L−1. Though GC/MS-MS displays a ten-fold higher sensitivity than single quadrupole GC/MS, the precision of both methods in the concentration range was similar. LC/MS-MS has the advantage of no further sample preparation due to direct injection and leads for methylanilines and meta-, para-substituted chloroanilines to results sufficiently equivalent to the standardised GC/MS method. However, LC/MS-MS is not suitable for ortho-chloroaniline derivatives due to significantly lower ion yields than meta- and para-substituted chloroanilines.

Determination of Saccharides in Honey Using Supercritical Fluid Chromatography Coupled with Single Quadrupole Mass Spectrometry.

Saccharides are one of the most important biomoleculars in the world, which are responsible for various roles in living organisms. In this work, a combined use of mass spectrometry (MS) together with supercritical fluid chromatography (SFC) was applied for the separation of eight saccharides. A satisfactory separation was achieved within 15min on a Unitary Diol column using 10mM ammonium acetate in methanol as mobile phase additive at a temperature of 60°C. Compared to liquid chromatography-MS (LC-MS), the SFC-MS presented faster running time and better selectivity. The SFC-MS was validated and applied to the analysis of three saccharides (fructose, glucose and sucrose) present in different honey samples. The limits of detection (LODs) and limits of quantification (LOQs) of SFC-MS method for fructose, glucose and sucrose were 5.2, 4.5, 7.4μg/mL and 17.2, 14.9, 24.1μg/mL, respectively. The relative standard deviations for intrabatch and interbatch precision were <2.5% and the recoveries ranged from 97.6% to 103.1% for three concentration levels. The values of LODs and LOQs using SFC-MS method were lower (1.5-2.4-fold) than using LC-MS method. The results demonstrated the potential of SFC-MS for fast and sensitive determination of saccharides in honey.

Supercritical fluid extraction–supercritical fluid chromatography of saliva: Single‐quadrupole mass spectrometry monitoring of caffeine for gastric emptying studies†

Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2 =0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

Novel sampling strategy for alive animal volatolome extraction combined with GC-MS based untargeted metabolomics: Identifying mouse pup pheromones

In this study, we identify 11 mouse pup volatiles putatively involved in maternal care induction in adult females. For this purpose, we have adapted the dynamic headspace methodology to extract the volatolome of whole alive animals. Untargeted metabolomic methodology was used to compare the volatolome of neonatal (4–6 days) with elder pups until the age of weaning (21–23 days old). Pup volatolome was analyzed by gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) using automated thermal desorption for sample introduction. After data processing and multivariate statistical analysis, comparison with NIST spectral library allowed identifying compounds secreted preferentially by neonatal pups: di(propylen glycol) methyl ether, 4-nonenal, di(ethylene glycol) monobutyl ether, 2-phenoxyethanol, isomethyl ionone, tridecanal, 1,3-diethylbenzene, 1,2,4,5-tetramethylbenzene, 2-ethyl-p-xylene and tri(propylene glycol) methyl ether. Palmitic acid was enriched in the volatolome of fourth week youngsters compared to neonatal pups. The results demonstrated the great potential of the new sampling procedure combined with GC-MS based untargeted volatolomics to identify volatile pheromones in mammals.

Single Quadrupole Multiple Fragment Ion Monitoring Quantitative Mass Spectrometry.

Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.

Off-line two-dimensional liquid chromatography separation for the quality control of saponins samples from Quillaja Saponaria.

Quil-A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil-A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two-dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off-line configuration using basic instrumentation was promoted. Hence, reversed-phase liquid chromatography × reversed-phase liquid chromatography and hydrophilic interaction chromatography × reversed-phase liquid chromatography methods with ultraviolet and single-quadrupole mass spectrometry detection were kinetically optimized. The reversed-phase liquid chromatography × reversed-phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed-phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed-phase liquid chromatography × reversed-phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed-phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.