A fully automatic cross used solid-phase extraction online coupled with ultra-high performance liquid chromatography-tandem mass spectrometry system for the trace analysis of multi-class pharmaceuticals in water samples.

A fully automatic system, which integrated cross used solid-phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry, was developed and validated for the simultaneous determination of multi-class pharmaceuticals (62 in total) in Milli-Q water, tap water, lake water, and ground water. The online system allowed the cross-utilization of two SPE columns without significant carryover and achieved an automatic, sensitive and fast analysis, requiring about 14 min per analysis. The features of the online system were systematically investigated and the analytical conditions were fully optimized. Sixty-two pharmaceuticals were divided into two groups (acidic and basic) under different extraction conditions to increase the extraction efficiency. Under optimal conditions, all the correlation coefficients were greater than 0.9929. The LODs and the LOQs were in the range of 0.00119-0.623 ng L-1 and 0.00475-2.49 ng L-1, respectively. The RSDs% for the intra-/inter-day precision were less than 10.6% and 15.6%, respectively. The system recoveries ranged from 80.7 to 119.9%. Compared with the offline SPE method, the online cross used SPE-UHPLC-MS/MS method obtained higher sensitivity and reduced manual operations. Compared with the existing online SPE systems, this system can reduce the time per analysis. Finally, this online system was applied to the analyses of three real water samples. Based on the results, the online cross used SPE-UHPLC-MS/MS system as an automatic, sensitive and efficient technique showed great promise for the future in the trace analysis of multi-class pharmaceuticals in complex aqueous samples.

Quantitative determination of bioactive proteins in diphtheria tetanus acellular pertussis (DTaP) vaccine by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed to determine simultaneously the bioactive proteins including pertussis toxin (PT) subunits, filamentous hemagglutinin (FHA), pertactin (PRN) and fimbriae (FIM) in diphtheria, tetanus and acellular pertussis combined vaccine (DTaP). The trypsin digestion conditions were investigated in detail using PT reference to achieve satisfactory results in detection of the peptides on LC-MS/MS with a Bio-C18 column. The performance of the described method was evaluated using reference proteins and the results showed a wide linear range (0.15-24 ng µL-1), a high sensitivity (0.038 ng. µL-1 for FHA) and a good precision (RSD of peak area <3.3%). This novel LC-MS/MS method was applied to determine PT subunits, FHA, PRN and FIM in DTaP vaccines, a total of ten batches, obtained from five manufacturers. The results revealed clearly that batch-to-batch consistency of the DTaP vaccines in terms of the protein amounts was stable, while those from manufacturers were varied significantly. On the other hand, the amount of bioactive proteins in component DTaP vaccines was generally higher than those in co-purified DTaP vaccines. The described LC-MS/MS method was compared with Chinese Pharmacopeia method (Lowry method) and it was found that FHA and PRN amounts measured by the two methods were in good agreement. The LC-MS/MS method could provide the amounts of PT subunits. However, the Lowry method could not differentiate the subunits. The LC-MS/MS method was not only more selective and sensitive, but it can be used to determine simultaneously different bioactive proteins in complex matrix-formulated vaccines. The method was extended successfully in other purposes, such as the effect of detoxification on bioactive proteins and characterization of PT references from four organizations worldwide.

[Determination of tracheal cytotoxin in pertussis and diphtheria tetanus acellular pertussis vaccines using liquid chromatography-tandem mass spectrometry].

Tracheal cytotoxin (TCT) is a toxic glycopeptide, which contribute to the adverse effects of pertussis toxin (PT) and related vaccines. Although pharmacopeias limit the amount of TCT in PT product, there is no recommended TCT determination method in any pharmacopeia. In this study, a liquid chromatography-tandem mass spectrometry method was developed to determine TCT. Chromatographic conditions, including column-type and mobile-phase composition, were optimized. According to the literature reports, both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) can provide a good retention for TCT. A large amount of organic solvent is usually used for protein precipitation, which may affect the RPLC mode, leading to peak distortion, while such effects were not observed in HILIC mode. Thus, HILIC mode was used to analyze TCT in this study. The developed method had a wide linear range (5.76-369 ng/L), good precision (no more than 3.9%), satisfied recoveries in various matrices (96.4%-102.5%). The limit of quantification (LOQ) of the developed method was 1279 times lower than the one required by Chinese Pharmacopeia, wherein the required amount of TCT should be less than 2 pmol per dose. The developed method was used to detect TCT in pertussis vaccine (acellular component), pertussis vaccine (acellular, co-purified), co-purified diphtheria tetanus pertussis vaccine, and component diphtheria tetanus acellular pertussis vaccine. As a result, TCT was not detected in any of the selected samples indicating the safety of these vaccines and PT products.

A novel two-dimensional liquid chromatography - Mass spectrometry method for direct drug impurity identification from HPLC eluent containing ion-pairing reagent in mobile phases.

In this study, a novel two dimensional liquid chromatography - mass spectrometry (2D-LC-MS) method with use of a weak anion exchange column between the 1st DLC RP column and the 2nd DLC RP column (RP1-WAX-RP2) was developed and applied to identify drug impurities from MS incompatible mobile phases containing sodium 1-octanesulfonate and non-volatile buffer. The 1st DLC conditions follow exactly the original standard HPLC method recorded in Chinese Pharmacopeia (ChP), European Pharmacopeia (EP) or US Pharmacopeia (USP). An impurity fraction was collected with a built-in sample loop (100 µL) and transferred to the WAX column where 1-octanesulfonate and phosphate were trapped and removed. While, the impurity and other cations were eluted to the 2nd D column (RP2) for separation and identification by connected IT-TOF MS. Methods were programmed and applied to identify impurities in two generic drugs, sulpiride (hydrophilic drug with logP 0.57) and dobutamine (hydrophobic drug with logP 3.6). The results indicate that the methods based on RP1-WAX-RP2 column configuration offer a feasible solution for direct impurity identification in generic drug product or API without needs of off-line desalting from the MS incompatible mobile phases containing ion-pairing reagent and non-volatile buffer.

Lipidomics study of the protective effects of isosteviol sodium on stroke rats using ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry.

Isosteviol sodium (STV-Na) was reported to possess significant protective effects on ischemic stroke in recent years. However, the protective mechanism of STV-Na against stroke was still unclear. In this work, an untargeted lipidomics approach based on the ultra high-performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry (UHSFC-IT-TOF/MS) was employed to investigate the lipid profiles of stroke rats with STV-Na treatment for the first time. The possible mechanism of STV-Na was further elucidated. The UHSFC-IT-TOF/MS-based method achieved a fast separation of various lipids within 9 min with a qualified repeatability. Multivariate statistical analysis was used to show differences in lipid profiles induced by stroke and STV-Na treatment. The results showed a clear separation of the model group and the sham group, with the STV-Na group as well as EDA group located much closer to the sham group than the model group, which was consistent with the results of physiological and pathological assays, indicating the protective effects of STV-Na. Fifteen differential lipids that presented significant differences between the sham group and the model group were screened and identified. With the treatment of STV-Na, 15 differential lipids in stroke rats showed a tendency to the normal levels. Among them, 6 lipids were significantly reversed to the normal levels by STV-Na. The results of pathway analysis suggested the protective effects of STV-Na might be related to the regulation of several metabolic pathways including glycerophospholipid metabolism, arachidonic acid metabolism and sphingolipid metabolism. This work demonstrated that the UHSFC-IT-TOF/MS-based lipidomics profiling method was a useful tool to investigate the protective effects of STV-Na against stroke.

Determination of major aromatic constituents in vanilla using an on-line supercritical fluid extraction coupled with supercritical fluid chromatography.

An on-line supercritical fluid extraction coupled with supercritical fluid chromatography method was developed for the determination of four major aromatic constituents in vanilla. The parameters of supercritical fluid extraction were systematically investigated using single factor optimization experiments and response surface methodology by a Box-Behnken design. The modifier ratio, split ratio, and the extraction temperature and pressure were the major parameters which have significant effects on the extraction. While the static extraction time, dynamic extraction time, and recycle time had little influence on the compounds with low polarity. Under the optimized conditions, the relative extraction efficiencies of all the constituents reached 89.0-95.1%. The limits of quantification were in the range of 1.123-4.747 µg. The limits of detection were in the range of 0.3368-1.424 µg. The recoveries of the four analytes were in the range of 76.1-88.9%. The relative standard deviations of intra- and interday precision ranged from 4.2 to 7.6%. Compared with other off-line methods, the present method obtained higher extraction yields for all four aromatic constituents. Finally, this method has been applied to the analysis of vanilla from different sources. On the basis of the results, the on-line supercritical fluid extraction-supercritical fluid chromatography method shows great promise in the analysis of aromatic constituents in natural products.

Automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma.

An automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim-pack MAYI-C8 (G) column was used as a solid-phase extraction column, and all the analytes were separated on a Shim-pack XR-ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5 mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column-head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321-2.75 µg/L and the recoveries ranged from 75.9 to 122%. Compared with the off-line ultra-high performance liquid chromatography and the reported methods, this validated on-line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on-line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.

Pre-column dilution large volume injection ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of multi-class pesticides in cabbages.

Pre-column dilution large volume injection (PD-LVI), a novel sample injection technique for reverse phase ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed in this study. The PD-LVI UHPLC-MS/MS system was designed by slightly modifying the commercial UHPLC-MS/MS equipment with a mixer chamber. During the procedure of PD-LVI, sample solution of 200µL was directly carried by the organic mobile phase to the mixer and diluted with the aqueous mobile phase. After the mixture was introduced to the UHPLC column in a mobile phase of acetonitrile-water (15/85, v/v), the target analytes were stacked on the head of the column until following separation. Using QuEChERS extraction, no additional steps such as solvent evaporation or residue redissolution were needed before injection. The features of PD-LVI UHPLC-MS/MS system were systematically investigated, including the injection volume, the mixer volume, the precondition time and the gradient elution. The efficiency of this approach was demonstrated by direct analysis of 24 pesticides in cabbages. Under the optimized conditions, low limits of detection (0.00074-0.8 ng/kg) were obtained. The recoveries were in the range of 63.3-109% with relative standard deviations less than 8.1%. Compared with common UHPLC-MS/MS technique, PD-LVI UHPLC-MS/MS showed significant advantages such as excellent sensitivity and reliability. The mechanism of PD-LVI was demonstrated to be based on the column-head stacking effect with pre-column dilution. Based on the results, PD-LVI as a simple and effective sample injection technique of reverse phase UHPLC-MS/MS for the analysis of trace analytes in complex samples showed a great promising prospect.

An automatic versatile system integrating solid-phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry using a dual-dilution strategy for direct analysis of auxins in plant extracts.

An automatic versatile system which integrated solid phase extraction (SPE) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Diverse commercial SPE columns can be used under an ambient pressure in this online system realized by a dual-dilution strategy. The first dilution enabled the direct injection of complex samples with minimal pretreatment, and the second dilution realized direct introduction of large volume of strong eluent into the UHPLC column without causing peak broadening or distortion. In addition, a post-column compensation mode was also designed for the matrix-effects evaluation. The features of the online system were systematically investigated, including the dilution effect, the capture of desorption solution, the column-head stacking effect and the system recovery. Compared with the offline UHPLC system, this online system showed significant advantages such as larger injection volume, higher sensitivity, shorter analysis time and better repeatability. The feasibility of the system was demonstrated by the direct analysis of three auxins from different plant tissues, including leaves of Dracaena sanderiana, buds and petals of Bauhinia. Under the optimized conditions, the whole analysis procedure took only 7min. All the correlation coefficients were greater than 0.9987, the limits of detection and the limits of quantitation were in the range of 0.560-0.800ng/g and 1.80-2.60ng/g, respectively. The recoveries of the real samples ranged from 61.0 to 117%. Finally, the post-column compensation mode was applied and no matrix-effects were observed under the analysis conditions. The automatic versatile system was rapid, sensitive and reliable. We expect this system could be extended to other target analytes in complex samples utilizing diverse SPE columns.

[Determination of 14 heterocyclic aromatic amines in wine by liquid chromatography-ion trap-time of flight tandem mass spectrometry].

A rapid qualitative and quantitative analytical method was developed for the simultaneous determination of 14 heterocyclic aromatic amines (HAAs) in wine by liquid chromatography-ion trap-time of flight tandem mass spectrometry (LC-IT-TOF MS). HAAs were extracted from the samples by ethyl acetate under alkaline condition. The quantitation was carried out using internal standard method. The separation of HAAs was carried out based on Phenomenex Kinetex C18 100A column (100 mm x 2.1 mm, 2.6 microm), with a gradient elution of acetonitrile and 30 mmol/L ammonium formate at a flow rate of 0.4 mL/min. The analytes were detected under positive-ion electrospray ionization mode. The results showed that the linear ranges of the 14 HAAs were 1-500 microg/L with limits of detection (signal/noise = 3) of 0.33-1.77 microg/L. The average recoveries of all the compounds spiked in wine samples at three levels of 10, 50, 100 microg/L were in the ranges of 71.6%-96.4%, 72.9%-101.9%, 74.5%-103.3%, with the corresponding relative standard deviations (RSDs, n = 6) of 2.9%-7.9%, 1.7%-5.3%, 1.8%-4.8%, respectively. The established method is simple, rapid, accurate, and has wide linear range and high sensitivity. It can be applied to the simultaneous analysis of the HAAs in wine.

[Rapid screening and confirmation of hormones in health foods by high performance liquid chromatography-ion trap-time of flight tandem mass spectrometry].

A method was developed for the screening, confirmation and quantification of 21 hormones including progesterones, etrogens, glucocorticoids and resorcylic acid lactones in health foods by high performance liquid chromatography-ion trap-time of flight tandem mass spectrometry (HPLC-IT-TOF-MS). The analytes in the sample were extracted with acetonitrile containing 1.0% (v/v) acetic acid and the extract was purified with the mixed QuEChERS sorbents. In the chromatographic analysis, the 21 target compounds were separated on a C18 column with the gradient elution using the mobile phases of acetonitrile and water containing 0.1% acetic acid. The mass analyzer was performed in positive and negative full scan modes in one injection at the same time. The results showed that the linear ranges of the 21 hormones were 5.0-250 microg/L with the correlation coefficients above 0.993 and the limits of quantification (LOQ, S/N > or = 10) were 2.0-5.0 microg/kg and 1.0-2.5 microg/L for capsule and oral solution, respectively. The method validation was carried out at three spiked levels, and the recoveries were 60.2%-116.0% with the relative standard deviations (RSDs) of 7.0%-18.3%. The screening of analytes was performed by precision mass matching and library searching. The secondary fragment ion spectra were employed to the confirmation. This method is simple, fast, reliable, and can be applied to the simultaneous screening and determination of hormones in health foods.

Integration of high accuracy N-terminus identification in peptide sequencing and comparative protein analysis via isothiocyanate-based isotope labeling reagent with ESI ion-trap TOF MS.

A multifunctional isothiocyanate-based isotope labeling reagent, [d (0)]-/[d (6)]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b(1) ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b(1) ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.

Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time-of-flight-mass spectrometry.

We herein present a chemical profiling method to efficiently process the information acquired by ultra fast liquid chromatography (UFLC)-electrospray ionization source in combination with hybrid ion trap and high-resolution time-of-flight mass spectrometry (UFLC-(ESI)-IT-TOF/MS), facilitating the structural determination of serial components contained in crude or processed traditional Chinese medicine (TCM). Under the optimized UFLC and IT-TOF-MS(n) conditions, over 39 compounds were separated and detected in crude or processed Fructus corni within 25 min. The components were identified by comparing the mass spectra and retention time with reference compounds, or tentatively assigned by elucidating low-energy collision-induced dissociation (CID) fragment ions and matching empirical molecular formula with that of the published compounds. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used to elucidate the processed mechanism of F. corni, which is regularly affected by the processing conditions. This study provides a novel approach and methodology to identify the complicated components from various complex mixtures such as crude TCM, processed TCM, and biological samples. It can be used as a valid analytical method for further understanding the processing mechanism of TCM, along with the intrinsic quality control of TCM and its processed product.

Characterization and sequence identification of angiotensin II by a novel method involving ultra-fast liquid chromatography assay coupled with matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight five tandem mass spectrometry analysis.

High-throughput proteomics aims to investigate dynamically changing proteins expressed by a full organism, specific tissue or cellular compartment under certain conditions. High-sensitivity mass spectrometry has gradually become a significant tool for characterizing peptides. Here, we analyzed angiotensin II using ultra-fast liquid chromatography (UFLC) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). First, we applied UFLC in isolating and collecting the angiotensin II, and then Axima-Resonance (MALDI-QIT-ToF MS(5)) was adopted, which enables collision-induced dissociation-MS(5) analysis for fine structural characterization of angiotensin II. Resultant MS, MS(2), MS(3) and MS(4) spectra of interested [M+H](+) ions selected as precursor ions yielded detailed information about the sites of fragmentation as well as the amino acid sequence for angiotensin II; meanwhile, the average deviation between theoretical mass and actually measured mass from MS to MS(5) spectra was only 0.32 Da. It indicated that Axima-Resonance was capable of analyzing the peptide sequence accurately and provide the corresponding fragmentation information thoroughly, thus suggesting a potential strategy involving UFLC assay coupled with MALDI-QIT-ToF MS(5) analysis on high-throughput proteomics study in future.