Quantitative method for analysis of tobacco‐specific N‐nitrosamines in mainstream cigarette smoke by using heart‐cutting two‐dimensional liquid chromatography with tandem mass spectrometry

A heart‐cutting two dimensional liquid chromatography coupled with tandem mass spectrometry method was developed for the analysis of tobacco‐specific N‐nitrosamines (TSNAs) at low concentration level in Virginia‐type cigarette smoke. A strong cation exchange column was utilized for the first dimensional separation, which effectively removed acidic and neutral components in the smoke, followed by a reversed phase liquid chromatography coupled with tandem mass spectrometric analysis. To capture components of the TSNAs in the effluent on the trapping column, a compensating pump was applied for online dilution and pH adjustment during the period of the TSNAs fraction transferring and enrichment. Highly sensitive determination of the TSNAs in mainstream cigarette smoke was achieved by isotope deuterated internal standards under the multiple reaction monitoring mode. Compared with traditional methodologies, the method was almost no matrix interference. Limits of quantity for the TSNAs were within 0.027–0.094 ng/mL, and the results showed good reproducibility and accuracy. Finally, the new method was applied for analysis of the Kentucky reference cigarettes and the results agreed well with joint experiments of Cooperation Centre for Scientific Research Relative to Tobacco.     

The use of online heart‐cutting high‐performance liquid chromatography coupled with linear ion trap mass spectrometry in the identification of impurities in vidarabine monophosphate

It is difficult to identify unknown impurities in nucleotide analogues by mass spectrometry because mass‐spectrometry‐incompatible mobile phases need to be used to separate the major ingredient from impurities. In this study, vidarabine monophosphate was selected, and unknown impurities were identified by online heart‐cutting two‐dimensional high‐performance liquid chromatography and linear ion trap mass spectrometry. The one‐dimensional reversed‐phase column was filled with a mobile phase containing nonvolatile salt. In two‐dimensional high‐performance liquid chromatography, we used an Acclaim Q1 column with volatile salt, and the detection wavelength was 260 nm. The mass spectrum was scanned in positive‐ and negative‐ion mode. The online heart‐cutting and online demineralization technique ensured that the mobile phase was compatible with mass spectrometry; seven impurities were identified by MS² and MS³ fragments. The mass fragmentation patterns of these impurities were investigated. The two isomers were semiprepared and complemented by nuclear magnetic resonance. The results were further compared with those of normal‐phase high‐performance liquid chromatography with mass spectrometry. The online heart‐cutting two‐dimensional high‐performance liquid chromatography with mass spectrometry was superior in identifying more impurities. The method solves the problem of incompatibility between the mobile phase and mass spectrometry, so it is suitable for identifying unknown impurities. This method may also be used for investigating impurities in other nucleotide analogues.     

Two‐dimensional solid‐phase extraction strategy for the selective enrichment of aminoglycosides in milk

An orthogonal two‐dimensional solid‐phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed‐phase liquid chromatography material (C₁₈) and a weak cation‐exchange material (TGA) were integrated in a single solid‐phase extraction cartridge. The feasibility of two‐dimensional clean‐up procedure that experienced two‐step adsorption, two‐step rinsing, and two‐step elution was systematically investigated. Based on the orthogonality of reversed‐phase and weak cation‐exchange procedures, the two‐dimensional solid‐phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed‐phase solid‐phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation‐exchange solid‐phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two‐dimensional clean‐up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk.     

Visualization and application of amino acid retention coefficients obtained from modeling of peptide retention

We introduce a method for data inspection in liquid separations of peptides using amino acid retention coefficients and their relative change across experiments. Our method allows for the direct comparison between actual experimental conditions, regardless of sample content and without the use of internal standards. The modeling uses linear regression of peptide retention time as a function of amino acid composition. We demonstrate the pH dependency of the model in a control experiment where the pH of the mobile phase was changed in controlled way. We introduce a score to identify the false discovery rate on peptide spectrum match level that corresponds to the set of most robust models, i.e. to maximize the shared agreement between experiments. We demonstrate the method utility in reversed‐phase liquid chromatography using 24 datasets with minimal peptide overlap. We apply our method on datasets obtained from a public repository representing various separation designs, including one‐dimensional reversed‐phase liquid chromatography followed by tandem mass spectrometry, and two‐dimensional online strong cation exchange coupled to reversed‐phase liquid chromatography followed by tandem mass spectrometry, and highlight new insights. Our method provides a simple yet powerful way to inspect data quality, in particular for multidimensional separations, improving comparability of data at no additional experimental cost.     

Anti‐inflammatory bioactive equivalence of combinatorial components β‐carboline alkaloids identified in Arenaria kansuensis by two‐dimensional chromatography and solid‐phase extraction coupled with liquid–liquid extraction enrichment technology

Bioactive equivalent combinatorial components play a critical role in herbal medicines. However, how to discover and enrich them efficiently is a question for herbal pharmaceuticals researchers. In our work, a novel two‐dimensional reversed‐phase/hydrophilic interaction high‐performance liquid chromatography method was established to perform real‐time components trapping and combining for preparation and isolation of coeluting components. Arenaria kansuensis was taken as an example, and solid‐phase extraction coupled with liquid–liquid extraction as a simple and efficient method for enriching trace components, reversed phase column coupled with hydrophilic interaction liquid chromatography XAmide column as two‐dimensional chromatography technology for isolation and preparation of coeluting constituents, enzyme‐linked immune‐sorbent assay as bio‐guided assay, and anti‐inflammatory bioactivity evaluation for bioactive constituents. A combination of 12 β‐carboline alkaloids was identified as anti‐inflammatory bioactive equivalent combinatorial components from A. kansuensis , which accounts for 1.9% w/w of original A. kansuensis . This work answers the key question of which are real anti‐inflammatory components from A. kansuensis and provides a fast and efficient approach for discovering and enriching trace β‐carboline alkaloids from herbal medicines for the first time. More importantly, the discovery of bioactive equivalent combinatorial components could improve the quality control of herbal products and inspire a herbal medicine based on combinatorial therapeutics.     

Development and Validation of a Simple and Efficient Method for the Determination of Pendimethalin and Its Metabolite M455H001 in Soil by Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS)

A new analytical method has been developed and described for the rapid determination of pendimethalin and its major metabolite M455H001 in soil by liquid chromatography coupled with ion-spray tandem mass spectrometry (LC-MS/MS) after a single acidic solvent extraction. The chromatographic separation of the analytes was achieved using a Zorbax C18 reversed phase column and water/0.1% formic acid and methanol as the mobile phase at a flow rate of 0.2?mL?min^?1. The recoveries of the method ranged from 78.8% to 119.8% for pendimethalin and from 73.7% to 108.8% for M455H001, and the relative standard deviation was lower than 16% for both analytes. The validated limit of quantification was 0.01?µg?g^?1 soil dry weight for both compounds. The matrix effects were evaluated and were less than 20% for both substances in the examined soil samples. It is concluded that the method is easy, with reasonable consumption of reagents, characterized by reliability and sensitivity, and therefore, it is suitable for monitoring the levels of pendimethalin and its major metabolite M455H001 in soils.     

Free amino acids analysis by liquid chromatography with tandem mass spectrometry in several botanicals with antioxidant character

A novel method based on liquid chromatography with tandem mass spectrometry for the analysis of 19 amino acids in plant materials is described. For the analysis, the plant material is extracted with 0.1 N hydrochloric acid with internal standards present in the extraction solution. The filtered extracts are injected using no clean‐up into the liquid chromatographic system coupled with a triple‐quadrupole tandem mass spectrometer with an electrospray ionization source. The analytes are separated using ion pair chromatography on a reversed‐phase column. The detection is performed in multiple‐reaction monitoring positive‐ion mode. Quantitation is obtained using calibrations. The validated procedure has been applied for the analysis of amino acids in 18 samples of plant material including botanicals with antioxidant character. The analysis requires 16 min separation time, has excellent precision and accuracy allowing amino acid analysis in a wide range of concentrations.     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

Determination of organophosphate esters in water samples by mixed‐mode liquid chromatography and tandem mass spectrometry

A simple and sensitive method for the determination of organophosphate esters in water samples by mixed‐mode liquid chromatography with electrospray ionization tandem mass spectrometry coupled with solid‐phase extraction is developed. Using seven alkyl phosphates, three chlorinated alkyl phosphates, and four aryl phosphates as the targets, the developed method was systematically evaluated on the basis of the influence of the solid‐phase extraction cartridge, eluting solvent, sample‐loading volume, mobile phase condition, and the separation of reversed‐phase chromatography and mixed‐mode liquid chromatography. Under the optimal conditions, these organophosphate esters can be extracted by ENVI‐18 cartridge, eluted by 6 mL of 25% dichloromethane in acetonitrile, and then qualified and quantified by mixed‐mode liquid chromatography with tandem mass spectrometry in the multiple reaction‐monitoring mode. The application of mixed‐mode liquid chromatography endows the separation with reasonable retention for both hydrophilic and hydrophobic organophosphate esters regardless of their polarity, which is hardly achieved by reversed‐phase chromatography. Good linearity (from 0.9877 to 0.9969), low quantification limits (1–35 ng/L after extraction of 100 mL of river water), and acceptable recovery rates (58.6–116.2%, with the relative standard deviation <18.0%) were obtained. Finally, the established method was used for analyzing surface water samples, and the good applicability of this method was demonstrated.     

Separation of selenium species and their sensitive determination in rice samples by ion‐pairing reversed‐phase liquid chromatography with inductively coupled plasma tandem mass spectrometry

A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion‐pairing reversed‐phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion‐exchange chromatography and ion‐pairing reversed‐phase chromatography was performed. The results indicated that ion‐pairing reversed‐phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02–0.12 μg/L, and recoveries of five selenium species were 75–114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium‐enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.     

Development of an improved online comprehensive hydrophilic interaction chromatography × reversed‐phase ultra‐high‐pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis

In this study, an improved online comprehensive two‐dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 μm, cross‐linked diol) was employed in the first dimension, while a reversed‐phase column based on monodisperse sub‐2 μm fully porous particles (50 × 3.0 mm, 1.9 μm d.p.) with high surface area (410 m²/g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed‐phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop‐based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time‐of‐flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples.     

Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid‐phase extraction coupled with two‐dimensional preparative liquid chromatography

An offline preparative two‐dimensional reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid‐phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula . First, the non‐flavonoids were removed using an XAmide solid‐phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two‐dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula . Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials.     

In situ derivatization–liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl‐4‐hydroxyproline by liquid chromatography–tandem mass spectrometry

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography–mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water‐immiscible organic phase. In the present work, the derivatization–liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker l ‐prolyl‐4‐l ‐hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N‐isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP‐C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7‐PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization–liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography–tandem mass spectrometry in the complex urine matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Magnetic solid‐phase extraction of tobacco‐specific N‐nitrosamines using magnetic graphene composite as sorbent

Tobacco‐specific N‐nitrosamines are carcinogenic components in mainstream cigarette smoke. To explore tobacco‐specific N‐nitrosamine release levels in cigarettes, a magnetic solid‐phase extraction procedure using magnetic graphene composite as sorbent for fast enrichment of tobacco‐specific N‐nitrosamine was developed. Under optimal conditions, a tobacco‐specific N‐nitrosamine determination method was successfully proposed by combining magnetic solid‐phase extraction procedure and high‐performance liquid chromatography coupled with tandem mass spectrometry. The method's limit of detection for tobacco‐specific N‐nitrosamines in mainstream cigarette smoke ranged from 0.018 to 0.057 ng/cigarette. Good linearities were obtained with correlation coefficients above 0.9992. The accuracies of tobacco‐specific N‐nitrosamines in a spiked mainstream cigarette smoke sample were from 89.3 to 109.4%, with a relative standard deviation of less than 11.2%. The proposed method has the merits of rapidity and high sensitivity. Finally, the method was successfully applied to tobacco‐specific N‐nitrosamine analysis in real samples.     

Optimization of a high‐resolution radical scavenging assay coupled on‐line to reversed‐phase liquid chromatography for antioxidant detection in complex natural extracts

This paper reports the optimization of the on‐line coupling of 2,2′‐azinobis(3‐ethylbenzothiazoline)‐6‐sulfonic acid based radical scavenging assays with reversed‐phase high‐performance liquid chromatography. The residence time in the reactor was reduced to 6.4 s to ensure minimal peak broadening and loss of separation. Peak capacity losses between compound detection and measurement of the radical scavenging potential were reduced to 10% and lower on coupled column systems. The methodology was successfully applied for the detection of the scavenging activity of molecules encompassing a broad hydrophobicity range. The method shows promise for the assessment of low‐molecular‐weight polyphenols in red wine by coupled‐column high‐resolution high‐performance liquid chromatography with mass spectrometry analysis.     

Analysis of allergens in tubeimu saponin extracts by using rat basophilic leukemia 2H3 cell‐based affinity chromatography coupled to liquid chromatography and mass spectrometry

An affinity two‐dimensional chromatography method was developed for the recognition, separation, and identification of allergic components from tubeimu saponin extracts, a preparation often injected to treat various conditions as indicated by traditional Chinese medicine. Rat basophilic leukemia‐2H3 cell membranes were used as the stationary phase of a membrane affinity chromatography column to capture components with affinity for mast cells that could be involved in a degranulation reaction. The retained components were enriched and analyzed by membrane affinity chromatography with liquid chromatography and mass spectrometry via a port switch valve. Suitability and reliability of the method was investigated using appropriate standards, and then, the method was applied to identify components retained from tubeimu saponin extracts. Tubeimoside A was identified in this way as a potential allergen, and degranulation assays confirmed that tubeimoside A induces RBL‐2H3 cell degranulation in a dose‐dependent manner. An increase in Ca²⁺ influx indicated that degranulation induced by tubeimoside A is likely Ca²⁺ dependent. Coupled with the degranulation assay, RBL‐2H3 cell‐based affinity chromatography coupled with liquid chromatography and mass spectrometry is an effective method for screening and identifying allergic components from tubeimu saponin extracts.     

Determination of multi‐pesticide residue in tobacco using multi‐walled carbon nanotubes as a reversed‐dispersive solid‐phase extraction sorbent

A multi‐pesticide residue determination method based on a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method using multiwalled carbon nanotubes as reversed‐dispersive solid‐phase extraction material was validated in 37 representative pesticides in tobacco. Determination was performed using liquid chromatography with tandem mass spectrometry in multiple reaction monitoring mode. Three major types of tobacco leaf samples, namely, flue‐cured, burley, and oriental tobacco were studied and compared. Three factors (extraction time, external diameter, and amount of extraction material used) that could affect the performance of multi‐walled carbon nanotubes were investigated. Optimization of sample preparation and determination allowed recoveries between 70.8 and 114.8% for all 37 pesticides with < 20.0% relative standard deviations at three spiking levels of 20, 50, and 200 μg/kg. The limits of quantification and limits of detection for the 37 pesticides ranged within 0.46–28.57 and 0.14–8.57 μg/kg at a signal‐to‐noise ratio of 10 and 3, respectively.     

Comprehensive metabonomic analysis of heart tissue from isoproterenol‐induced myocardial infarction rat based on reversed‐phase and hydrophilic interaction chromatography coupled to mass spectrometry

We aim to describe the metabonomic characteristics of myocardial infarction rats. High‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was utilized to develop a metabonomic method of the heart homogenates of myocardial infarction rats. Hydrophilic interaction chromatography allows the analysis of high polar metabolites, providing complementary information to reversed‐phase liquid chromatography. We combined reversed phase and hydrophilic interaction chromatographic separations to analyze 18 samples, ten from myocardial infarction rat hearts and eight from normal rat hearts. A total of 16 potential biomarkers in rat heart tissue were screened out, primarily related to oxidative stress, nitric oxide damage, taurine, and hypotaurine metabolism and sphingolipid metabolism. This research showed that a comprehensive metabonomic study is a useful tool to reveal the underlying mechanism of myocardial infarction.