Hybrid Quadrupole Linear Ion Trap Research Articles

Off-Line SPE LC-LRMS Polyphenolic Fingerprinting and Chemometrics to Classify and Authenticate Spanish Honey.

The feasibility of non-targeted off-line SPE LC-LRMS polyphenolic fingerprints to address the classification and authentication of Spanish honey samples based on both botanical origin (blossom and honeydew honeys) and geographical production region was evaluated. With this aim, 136 honey samples belonging to different botanical varieties (multifloral and monofloral) obtained from different Spanish geographical regions with specific climatic conditions were analyzed. Polyphenolic compounds were extracted by off-line solid-phase extraction (SPE) using HLB (3 mL, 60 mg) cartridges. The obtained extracts were then analyzed by C18 reversed-phase LC coupled to low-resolution mass spectrometry in a hybrid quadrupole-linear ion trap mass analyzer and using electrospray in negative ionization mode. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were employed to assess the pattern recognition capabilities of the obtained fingerprints to address honey classification and authentication. In general, a good sample discrimination was accomplished by PLS-DA, being able to differentiate both blossom-honey and honeydew-honey samples according to botanical varieties. Multiclass predictions by cross-validation for the set of blossom-honey samples showed sensitivity, specificity, and classification ratios higher than 60%, 85%, and 87%, respectively. Better results were obtained for the set of honeydew-honey samples, exhibiting 100% sensitivity, specificity, and classification ratio values. The proposed fingerprints also demonstrated that they were good honey chemical descriptors to deal with climatic and geographical issues. Characteristic polyphenols of each botanical variety were tentatively identified by LC-MS/MS in multiple-reaction monitoring mode to propose possible honey markers for future experiments (i.e., naringin for orange/lemon blossom honeys, syringic acid in thyme honeys, or galangin in rosemary honeys).

Multiclass Comparative Analysis of Veterinary Drugs, Mycotoxins, and Pesticides in Bovine Milk by Ultrahigh-Performance Liquid Chromatography-Hybrid Quadrupole-Linear Ion Trap Mass Spectrometry.

A multiclass and multiresidue method for simultaneously screening and confirming veterinary drugs, mycotoxins, and pesticides in bovine milk was developed and validated with ultrahigh-performance liquid chromatography–hybrid quadrupole–linear ion trap mass spectrometry (UHPLC-Qtrap-MS). A total of 209 targeted contaminants were effectively extracted using an optimized QuEChERS method. Quantitative and qualitative confirmation were achieved simultaneously by multiple reaction monitoring–information-dependent acquisition–enhanced product ion (MRM-IDA-EPI) scan mode. The validation results exhibited a good sensitivity with the LOQs of 0.05–5 μg/kg, which was satisfactory for their MRLs in China or EU. The recoveries of in-house spiked samples were in the range of 51.20–129.76% with relative standard deviations (RSD) between replicates (n = 3) 0.82% and 19.76%. The test results of 140 milk samples from supermarkets and dairy farms in China showed that cloxacillin, aflatoxin M1, acetamiprid, and fipronil sulfone were found with lower concentrations. Combined with the residue results from the literature, penicillin G and cloxacillin (beta-lactams), enrofloxacin and ciprofloxacin (fluoroquinolones), and sulfamerazine (sulfonamides) were more frequently detected in different countries and need to receive more attention regarding their monitoring and control.

Hemp Chemotype Definition by Cannabinoids Characterization Using LC-ESI(+)-LTQ-FTICR MS and Infrared Multiphoton Dissociation

The development and application of advanced analytical methods for a comprehensive analysis of Cannabis sativa L. extracts plays a pivotal role in order to have a reliable evaluation of their chemotype definition to guarantee the efficacy and safety in pharmaceutical use. This paper deals with the qualitative and quantitative determination of cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), and cannabigerol (CBG) based on a liquid chromategraphy-mass spectrometry (LC-MS) method using electrospray ionization in positive mode (ESI+), coupled with a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). For the first time, structural information of phytocannabinoids is available upon precursor ions’ isolation within the FTICR trapping cell and subsequent fragmentation induced by infrared multiphoton dissociation (IRMPD). Such fragmentation and accurate mass measurement of product ions, alongside collision-induced dissociation (CID) within LTQ, was advantageous to propose a reliable fragmentation pattern for each compound. Then, the proposed LC-ESI(+)-LTQ-FTICR MS method was successfully applied to the hemp chemotype definition of three registered Italian accessions of hemp C. sativa plants (Carmagnola C.S., Carmagnola, and Eletta Campana), thus resulting in the Eletta Campana accession being the best one for cannabis product manufacturing.

A workflow for multiclass determination of 256 pesticides in essential oils by liquid chromatography tandem mass spectrometry using evaporation and dilution approaches: Application to lavandin, lemon and cypress essential oils

This paper describes the determination of 256 multiclass pesticides in cypress and lemon essential oils (EOs) by the way of liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS/MS) analysis using the scheduled selected reaction monitoring mode (sSRM) available on a hybrid quadrupole linear ion trap (QLIT) mass spectrometer. The performance of a sample preparation of lemon and cypress EOs based on dilution or evaporation under nitrogen assisted by a controlled heating were assessed. The best limits of quantification (LOQs) were achieved with the evaporation under nitrogen method giving LOQs≤10µgL−1 for 91% of the pesticides. In addition the very satisfactory results obtained for recovery, repeatability and linearity showed that for EOs of relatively low evaporation temperature, a sample preparation based on evaporation under nitrogen is well adapted and preferable to dilution. By compiling these results with those previously published by some of us on lavandin EO, we proposed a workflow dedicated to multiresidue determination of pesticides in various EOs by LC–ESI/sSRM. Among the steps involved in this workflow, the protocol related to mass spectrometry proposes an alternative confirmation method to the classical SRM ratio criteria based on a sSRM survey scan followed by an information-dependent acquisition using the sensitive enhanced product ion (EPI) scan to generate MS/MS spectra then compared to a reference. The submitted workflow was applied to the case of lemon EOs samples highlighting for the first time the simultaneous detection of 20 multiclass pesticides in one EO. Some pesticides showed very high concentration levels with amounts greatly exceeding the mgL−1.

Simultaneous Detection of Flavonoids, Phenolic Acids and Alkaloids in Abri Herba and Abri Mollis Herba using Liquid Chromatography Tandem Mass Spectrometry.

Abri Herba has remarkable properties, such as cleanup heat detoxification, dampness and activating blood circulation to dissipate blood stasis; as a result, it has been applied to treat acute or chronic hepatitis and mastitis. Abri mollis Herba is often used as Abri Herba. Hierarchical cluster analysis (HCA) was applied to compare the similarities and differences of the chemical compositions in the two types of medicinal materials. To establish a high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous analysis of 15 flavonoids, two phenolic acids and three alkaloids in Abri Herba and Abri mollis Herba. The chromatographic separation was performed on a C18 column with a mobile phase of methanol (A), acetonitrile (B) and 0.5‰ acetic acid in water (C) using gradient elution. The detection of the target compounds was performed in multiple-reaction monitoring (MRM) mode using a hybrid quadrupole linear ion trap mass spectrometer equipped with positive/negative ion-switching electrospray ionisation (ESI) source. The developed method is reliable, sensitive and specific. In addition, the method has been successfully applied to differentiate 15 batches of Abri Herba and 27 batches of Abri mollis Herba stems. Furthermore, a comparison of the contents among stems, roots and leaves from the same strain in seven batches of Abri mollis Herba and four batches of Abri Herba has also been performed. HPLC-MS/MS method is sensitive and selective and can be suitable for the reliable quality control of Abri mollis Herba and Abri Herba.

The identification of lobeglitazone metabolites in rat liver microsomes and the kinetics of the in vivo formation of the major metabolite M1 in rats

The objective of this study was to elucidate the chemical structure of the metabolites derived from lobeglitazone (LB) during its incubation with rat liver microsomes and to characterize the kinetics of formation of the major metabolite M1 in vivo. Using high performance liquid chromatography coupled with a hybrid quadrupole linear ion trap, the metabolites were derived from LB during its incubation with rat liver microsomes. From various fragmentation patterns obtained from the metabolites, LB was biotransformed into 5 metabolites in the incubation, in which demethylation and hydroxylation appeared to be the principle metabolic pathways in vitro; Amongst the five primary metabolites, M1, a demethylated derivative of LB, appeared to be the major metabolite of LB, based on a comparison on the peak intensities in the ion chromatogram. In a study of the in vivo kinetics of formation of M1 in rats, the rate of formation of M1 from LB was determined to be 0.252 and 0.216mL/min/kg at doses of 0.5mg/kg and 2mg/kg of LB, respectively, suggesting that the kinetics of M1 formation were linear in the dose range tested. Considering the fact that LB is primarily eliminated by hepatic metabolism in rats, the formation of M1 accounts for approximately 7.50–9.76% of the overall elimination of LB in rats.

Degradation and monitoring of acetamiprid, thiabendazole and their transformation products in an agro-food industry effluent during solar photo-Fenton treatment in a raceway pond reactor

In this study, pesticides acetamiprid and thiabendazole and their transformation products (TPs), seven from each pesticide, were successfully monitored during solar photo-Fenton treatment in a real secondary effluent from an agro-food industry spiked with 100μgL−1 of each pesticide. To this end, a highly sensitive procedure was developed, based on liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap mass spectrometry (QqLIT-MS). In addition, finding low-cost and operational technology for the application of AOPs would then facilitate their use on a commercial level. Simple and extensive photoreactors such as raceway pond reactors (RPRs) are therefore proposed as an alternative for the application of solar photo-Fenton. Results showed that high degradation could be achieved in a complex water matrix (>99% TBZ and 91% ACTM in 240min) using a 120-L RPR pilot plant as novel technology. The analyses indicated that after the treatment only three TPs from ACTM were still present in the effluent, while the others had been removed. The study showed that the goal of either just removing the parent compounds, or going one step further and removing all the TPs, can significantly change the treatment time, which would affect process costs.

Simultaneous quantification of 16 bioactive constituents in Common cnidium fruit by liquid chromatography–electrospray ionization-mass spectrometry

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 16 important bioactive constituents including nine coumarins, and seven flavonoids in Common cnidium fruit samples from different regions. The separation was performed on a C18 column with linear gradient elution of acetonitrile and 0.1% acetic acid at a flow rate of 1.0ml/min in 15min. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of 16 bioactive constituents was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by high performance liquid chromatography–tandem mass spectrometric method would be a well acceptable strategy to comprehensively control the quality of C. cnidium fruit.

Qualitative and quantitative determination of 15 main active constituents in Fructus Sophorae pill by liquid chromatography tandem mass spectrometry.

Background:Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill.Materials and Methods:Chromatographic separation was performed on a C18 column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes.Results:The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents.Conclusion:The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill.

Degradation of the cytostatic etoposide in chlorinated water by liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry: Identification and quantification of by-products in real water samples

Once discharged into the sewage system, many pharmaceuticals may undergo degradation reactions in the presence of chemical disinfectants, generating by-products that may possess enhanced toxicity relative to the parent compounds. For this reason, the stability of the widely used cytostatic etoposide in chlorinated water has been investigated for the first time in the present work. Taking advantage of the high-resolution/accurate-mass capabilities of the hybrid quadrupole-Orbitrap mass spectrometer Q Exactive, two new oxidation by-products of etoposide were reliably identified. The time course of etoposide and its by-products was followed at different pH values, free chlorine concentrations and water matrices. Finally, the occurrence of etoposide and its major identified by-product (3′-O-desmethyl etoposide) was investigated in real water samples by on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry using a 4000QTRAP hybrid quadrupole-linear ion trap mass spectrometer. The etoposide by-product was found in various river and wastewater samples at levels between 14 and 33ngL−1, whereas etoposide was not detected in any sample.

Pharmacokinetic and excretion study of three secoiridoid glycosides and three flavonoid glycosides in rat by LC-MS/MS after oral administration of the Swertia pseudochinensis extract.

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of swertiamarin, gentiopicroside, sweroside, mangiferin, isoorientin and isovitexin in rat plasma, bile, urine and feces after oral administration of Swertia pseudochinensis extract using sulfamethalazole (SMZ) as an internal standard (IS). The samples were pretreated and extracted by liquid-liquid extraction as the sample clean-up procedure. The chromatographic separation was performed on a C18 column with a linear gradient elution using a mobile phase consisted of 0.1% formic acid and methanol at a flow rate of 0.8 mL/min. The total run time was 10 min. Determination and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. A multiple-reaction monitoring scanning (MRM) method with electrospray ionization (ESI) source was employed with simultaneously monitoring the positive and negative electrospray ion source polarity in a single run. A full validation of the method was performed. The linearity of the analytical response was good, with correlation coefficients greater than 0.9953. The intra-day and inter-day precisions (RSD %) exhibited within 10.4%, and the accuracy (RE %) ranged from -8.7% to 9.5%. A non-compartmental model was employed to calculate the parameters. The values of elimination rate constants (Ke) ranged from 0.0026 to 0.0118 and the elimination half-life (T1/2) ranged from 58.4 to 263.0 min. This is the first report on pharmacokinetic and excretion study of Swertia pseudochinensis extract after oral administration. The results provided a meaningful basis for the clinical application of this herb.

Determination of eight pesticides of varying polarity in surface waters using solid phase extraction with multiwalled carbon nanotubes and liquid chromatography-linear ion trap mass spectrometry

We describe a MWCNT-based method for the solid-phase extraction of eight pesticides from environmental water samples. The analytes are extracted from 100 mL samples at pH 5.0 (containing 5 mmol L−1 of KCl) by passing the solution through a column filled with 20 mg of multiwalled carbon nanotubes. Following elution, the pesticides were determined by LC and electrospray ionization hybrid quadrupole linear ion trap MS. Two selected reaction monitoring transitions were monitored per compound, the most intense one being used for quantification and the second one for confirmation. In addition, an information-dependent acquisition experiment was performed for unequivocal confirmation of positive findings. Matrix effect was not found in real waters and therefore the quantitation was carried out with calibration graphs built with solvent based standards. Except for cymoxanil, the detection and quantitation limits in surface waters are in the range from 0.3 to 9.5 ng L−1 and 1.6 to 45.2 ng L−1, respectively. Recoveries from spiked ultrapure water are ~100 %, except for the most polar pesticides methomyl and cymoxanil. The same behavior is found for real water samples (except for phosalone). The relative standard deviation is <10 % in all cases.

Simultaneous determination of diclofenac, its human metabolites and microbial nitration/nitrosation transformation products in wastewaters by liquid chromatography/quadrupole-linear ion trap mass spectrometry

An analytical method was developed and validated for the first determination of five major human metabolites of the non-steroidal anti-inflammatory drug diclofenac as well as two microbial transformation products in wastewater. The method was based on the extraction of diclofenac and the chemically synthetized compounds by solid-phase extraction (SPE), using a hydrophilic–lipophilic balanced polymer followed by liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap mass spectrometry (QqLIT-MS). Quantitation was performed by the internal standard approach, to correct for matrix effects. The accuracy of the method was generally higher than 40% for raw and treated wastewater with a precision below 12%. In wastewater influent and effluent samples the detection limits for the majority of target compounds were 0.3–2.5ngL−1 and 0.1–3.1ngL−1, respectively. The method was applied to the analysis of influent and effluent wastewater samples from urban wastewater treatment plants. Moreover, to obtain an extra tool for confirmation and identification of the studied diclofenac-derived compounds, Information-Dependent Acquisition (IDA) experiments were performed, with selected reaction monitoring (SRM) as the survey scan and an enhanced product ion (EPI) scan as the dependent scan. Diclofenac and its major human metabolite, 4′-hydroxydiclofenac were detected in all samples at concentrations of 331–1150ngL−1 and 585–6000ngL−1, respectively. Neither microbial transformation product of diclofenac was detected in any of the influent samples analyzed, but in effluents, their concentrations ranged from 4 to 105ngL−1.

Quantitative determination of poly(amidoamine) dendrimers in urine by liquid chromatography/electrospray ionization hybrid quadrupole linear ion trap mass spectrometry

Dendrimer nanocarriers have become of increasing interest in the field of biomedicine for their drug delivery potential. Surface modifications and optimized nanosize control are the strategies being followed to enhance drug delivery efficacy and renal clearance, especially for dendrimers of a lower generation number. The aim of this study was the development and performance evaluation of an analytical method for the quantitative determination of polyamidoamine (PAMAM) dendrimers in urine. PAMAM dendrimers (generations G0 to G3) were analyzed using liquid chromatography/electrospray ionization hybrid quadrupole linear ion trap mass spectrometry (LC/ESI-QqLIT-MS). Quantitative analysis was performed in selected reaction monitoring (SRM) mode. To confer a higher degree of confidence on the identification of PAMAM dendrimers, an SRM scan and collision-induced dissociation (CID), as a dependent scan, were performed in one single run using the information-dependent acquisition (IDA) mode. The LC/ESI-QqLIT-MS method, in SRM mode, allowed quantitative determination in urine matrix with good repeatability and reproducibility (relative standard deviation (R.S.D.) from 2 to 15%), linearity (R >0.99) over the concentration range (6∙10-4 to 5∙10-2 mmol.L-1 ), and sensitivity within the micromolar range. The detection limit values were above 1∙10-4 mmol.L-1 in both solvent and urine, for the generations studied. The developed method has demonstrated a capability for the identification and quantification of PAMAM dendrimer nanoparticles in a complex liquid matrix. The use of an LC/ESI-QqLIT-MS system, of modest m/z range and unit resolution, offers an alternative in the analysis of lower generation PAMAM dendrimers between mass analyzers of higher resolution and the conventional LC-UV method that is commonly applied for dendrimer quantification, but which lacks sufficient identification capacity. Copyright © 2013 John Wiley & Sons, Ltd.

Simultaneous determination of four flavonoids and one phenolic acid in rat plasma by LC–MS/MS and its application to a pharmacokinetic study after oral administration of the Herba Desmodii Styracifolii extract

A sensitive and selective liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of four flavonoids (schaftoside, isovitexin, luteolin, and apigenin) and one phenolic acid (ferulic acid) in rat plasma using sulfamethoxazole as the internal standard (IS). The separation was performed using a Diamonsil C18 column, which was eluted with methanol (A) and 0.1‰ acetic acid (B). The gradient condition was as follows: 0-5min, 40-60% A; 5-6min, 60-95% A; and 6-10min, maintained at 95% A. The analytes were detected using a hybrid quadrupole linear ion trap mass spectrometer that was equipped with an electrospray ionization source in the negative ion and multiple-reaction monitoring modes. A full validation of the method was performed. The linearity of the analytical response was good, with correlation coefficients greater than 0.9925 for all of the compounds within the concentration range. The lower limits of quantitation (LLOQ) of schaftoside, isovitexin, luteolin, apigenin, and ferulic acid in rat plasma were 1.66, 0.84, 3.69, 1.70, and 3.91ng/mL, respectively. The intra-day and inter-day precisions of the investigated components exhibited an RSD within 13.20%, and the accuracy (RE%) ranged from -8.47% to 10.90%. The results indicated that the developed method is sufficiently reliable for the pharmacokinetic study of schaftoside, isovitexin, apigenin, luteolin, and ferulic acid in rats following oral administration of the Herba Desmodii Styracifolii extract.

QUANTITATIVE ANALYSIS OF TEN DITERPENOIDS IN RAT BILE AFTER ORAL ADMINISTRATION OF Isodon rubescens EXTRACT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY

Diterpenoids are the primary bioactive ingredients in Isodon rubescens named Dong Ling Cao in China which possess many pharmacological activities, including anti-tumor, anti-microbial, anti-inflammatory, anti-catastrophe, anti-oxidation, and anti-inflammatory effects. We employed a sensitive and selective high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) method for the quantification of ten diterpenoids in rat bile. The ten diterpenoids are effusanin A (1), lasiodonin (2), oridonin (3), epinodosin (4), nodosin (5), ponicidin (6), rabdoternin A (7), enmenol (8), lasiokaurin (9), and lasiokaurinol (10). The sulfamethoxazole was used as internal standard (IS). The bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). Chromatographic separation was performed on a C18 column with linear gradient elution using water and methanol at a flow rate of 0.8 mL/min. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode, respectively, using multiple–reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect, and several of stabilities have been validated for diterpenoids in rat bile samples. The results showed that this method is robust, specific, and sensitive and it can successfully fulfill the requirement of excretion study of the ten diterpenoids in Isodon rubescens.

Identification of cytochrome P450 isoenzymes involved in metabolism of (+)-praeruptorin A, a calcium channel blocker, by human liver microsomes using ultra high-performance liquid chromatography coupled with tandem mass spectrometry

Angular-type pyranocoumarins (APs) show attractive prospects in anti-hypertension, chemotherapy and anti-HIV treatment. Previous studies revealed extensive hepatic metabolisms of several APs following similar pathways. This study investigated the enzyme kinetics and the main CYP450 isozyme(s) involved in metabolism of (+)-praeruptorin A (dPA), an AP with significant cardio-protective activities, in human liver microsomes (HLMs) using ultra high-performance liquid chromatography coupled with a hybrid quadrupole-linear ion trap mass spectrometry (UHPLC–QT-MS/MS). dPA produced 6 metabolites via hydrolysis (M1–M3), oxidation (M4–M6), and hydrolysis followed by acyl migration (M2 or M3). Oxidation at the C-3′ side chain instead of the coumarin ring was consolidated with the aromatic proton signal in NMR spectra. The major metabolite (−)-cis-khellactone (M1) followed biphasic kinetics in HLMs with high affinity (Km1 0.02μM) and intrinsic clearance (CLint1, invitro1.29mL/min/mg protein), whereas other metabolites (M2–M6) fitted typical Michaelis–Menten kinetics with lower affinity (Km 3.85–39.13μM). Recombinant human CYP3A4 showed the highest activity toward M1 and M4 formation, while it was CYP2C19 for M2/M3 and M5 and CYP2B6 for M6. Principal component analysis of the metabolite formation profile of dPA also revealed the highest similarity between CYP3A4 and HLMs. Both quercetin (CYP2C8 inhibitor) and ketoconazole (CYP3A4 inhibitor) showed 60–100% inhibition of M1–M4 and M6 formations in HLMs, while M5 formation was mainly inhibited by α-naphthoflavone (CYP1A2 inhibitor, 70–80%) and quercetin (90%). Moreover, formations of all metabolites were predominantly inhibited by CYP3A4 antibody (37–68%). These findings shed a light on main involvement of CYP3A4 in human hepatic elimination of APs, indicating potential drug interactions.

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Simultaneous detection of flavonoids and phenolic acids in Herba Lysimachiae and Herba Desmodii Styracifolii using liquid chromatography tandem mass spectrometry

A sensitive liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous analysis of 15 flavonoids and 3 phenolic acids in Herba Lysimachiae and Herba Desmodii Styracifolii. Separation was performed using a Diamonsil C18 column, which was eluted with methanol (A) and 0.1‰ acetic acid (B). The gradient condition was as follows: 0–34min, 20–34% A; 34–38min, 34–95% A; maintained 95% A for the next 4min. Analytes were detected using a hybrid quadrupole linear ion trap mass spectrometer that was equipped with an electrospray ionisation source in the negative ion and multiple-reaction monitoring modes. A full validation of the method was performed, including the linearity, precision, and accuracy, as well as limits of detection and quantification. The results indicated that the developed method was simple, sensitive and reliable. Furthermore, the method was successfully applied to differentiate 16 batches of Herba Lysimachiae and 21 batches of Herba Desmodii Styracifolii.

Identification of ADP-ribosylation sites of CD38 mutants by precursor ion scanning mass spectrometry

Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases.