SpheriCal® -ESI: A dendrimer-based nine-point calibration solution ranging from m/z 273 to 1716 for electrospray ionization mass spectrometry peptide analysis.

RATIONALE: A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal® -ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use. METHODS: The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal® -ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na+ in the calibration solution, were evaluated. RESULTS: SpheriCal® -ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal® -ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na+ contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision-induced dissociation in both ion trap and tandem quadrupole mass spectrometers. CONCLUSIONS: SpheriCal® -ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing.

Standard substances free quantification makes LC/ESI/MS non-targeted screening of pesticides in cereals comparable between labs.

LC/ESI/MS is the technique of choice for qualitative and quantitative food monitoring; however, analysis of a large number of compounds is challenged by the availability of standard substances. The impediment of detection of food contaminants has been overcome by the suspect and non-targeted screening. Still, the results from one laboratory cannot be compared with the results of another laboratory as quantitative results are required for this purpose. Here we show that the results of the suspect and non-targeted screening for pesticides can be made quantitative with the aid of in silico predicted electrospray ionization efficiencies and this allows direct comparison of the results obtained in two different laboratories. For this purpose, six cereal matrices were spiked with 134 pesticides and analysed in two independent labs; a high correlation for the results with the R2 of 0.85.

The use of a double coaxial electrospray ionization sprayer improves the peak resolutions of anionic metabolites in capillary ion chromatography-mass spectrometry.

Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1-100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites.

Method comparison for N-glycan profiling: Towards the standardization of glycoanalytical technologies for cell line analysis.

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.

Preparation of Cyclic-1,N2-propano-2'-deoxyguanosine-d7 as an Internal Standard for ESI-MS/MS Determination of DNA Damage from Acetaldehyde.

Cyclic-1,N2-propano-2'-deoxyguanosine-d7 (CPr-dG-d7) was prepared as an isotopic internal standard (IS) for electrospray ionization tandem mass spectrometry (ESI-MS/MS) quantification of CPr-dG in DNA as a candidate cancer risk marker of acetaldehyde intake, mainly from drinking. The deuterated compound was reasonably synthesized from acetaldehyde-d4 and 2'-deoxyguanosine in deuterium oxide (D2O), preventing the deuterium atoms of acetaldehyde-d4 from being substituted by hydrogen atoms, which occurred seriously in aqueous synthesis media via keto-enol tautomerism. Furthermore, another deuterium atom was added from D2O to form CPr-dG-d7. After four weeks of storage in H2O at 10°C, CPr-dG-d7 was found to be sufficiently stable for practical use. The calibration curve of CPr-dG by using a hydrophilic interaction chromatography-ESI-MS/MS system with CPr-dG-d7 as the IS showed sufficient linearity from 1.0 × 10-10 to 4.0 × 10-9 M with r2 = 0.998.

An LC-MS/MS analysis for seven sex hormones in serum.

BACKGROUND: Comprehensive serum sex hormone profiling is essential for monitoring the occurrence and development of many related diseases. However, the current methods for multi-class sex hormone detection were always lack of Standard Reference Material (SRM) certification and suffered from large sample consumption. For improvement, we developed a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method focused on SRM certification and minimization of serum consumption for simultaneous quantification of seven mainstream serum sex hormones including estrogens (estrone E1, estradiol E2 and estriol E3), androgens (testosterone T, androstenedione AD, dehydroepiandrosterone DHEA) and progestogens (progesterone P). METHODS: To achieve one-batch analysis, a straightforward strategy was designed and carefully optimized. Schematically, serum was firstly spiked with isotope-labeled internal standards. Then, liquid-liquid extraction was performed with methyl tert-butyl ether. After drying under nitrogen, dansyl chloride was introduced for derivatization. Finally, the mixture was submitted to LC-MS/MS for quantification. RESULTS: The limit of quantification was 0.005 ng/mL for E1, E2 and E3, 0.01 ng/mL for T, P and AD, 0.25 ng/mL for DHEA. Inter- and intra-assay CVs were less than 11.8%. The selectivity was proved satisfactory by interference spiking tests. With systematical SRM validation, the mean bias of -5.4 to 4.7% was observed, which indicated excellent method reliability. We found significant positive bias in chemiluminescence immunoassay (CLIA) detection comparing with current method, which promoted us to reconsider our previous results on sex hormone regulation in male patients with coronary atherosclerotic disease. After redetecting the related samples, modified and improved conclusions were proposed. CONCLUSIONS: A LC-MS/MS method for multi-class serum sex hormone profiling was developed with SRM certification and minimized serum consumption. Taking advantages of such reliable method, the previous CLIA-based research findings on sex hormone regulation in male patients with coronary atherosclerosis were modified and improved after redetecting the same sample-pool.

Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Activated charcoal significantly improved the reliability of methods for quantitative analysis of endogenous substances in biological specimens: Glutathione and cysteine as cases.

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet "analyte-free authentic matrices" is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare "GSH&CYS-free biological matrix". The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples.

Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology.

Quantitative mass spectrometry imaging (MSI) is a robust approach that provides both quantitative and spatial information for drug candidates' research. However, because of complicated signal suppression and interference, acquiring accurate quantitative information from MSI data remains a challenge, especially for whole-body tissue sample. Ambient MSI techniques using spray-based ionization appear to be ideal for pharmaceutical quantitative MSI analysis. However, it is more challenging, as it involves almost no sample preparation and is more susceptible to ion suppression/enhancement. Herein, based on our developed air flow-assisted desorption electrospray ionization (AFADESI)-MSI technology, an ambient quantitative MSI method was introduced by integrating inkjet-printing technology with normalization of the signal extinction coefficient (SEC) using the target compound itself. The method utilized a single calibration curve to quantify multiple tissue types. Basic blue 7 and an antitumor drug candidate (S-(+)-deoxytylophorinidine, CAT) were chosen to initially validate the feasibility and reliability of the quantitative MSI method. Rat tissue sections (heart, kidney, and brain) administered with CAT was then analyzed. The quantitative MSI analysis results were cross-validated by LC-MS/MS analysis data of the same tissues. The consistency suggests that the approach is able to fast obtain the quantitative MSI data without introducing interference into the in-situ environment of the tissue sample, and is potential to provide a high-throughput, economical and reliable approach for drug discovery and development.

Liquid chromatography-tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma.

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Rapid methodology via mass spectrometry to quantify addition of soybean oil in extra virgin olive oil: A comparison with traditional methods adopted by food industry to identify fraud.

Fast and innovative methodology to monitors the addition of soybean oil in extra virgin olive oil was developed employing ESI-MS with ionization operating in positive mode. A certified extra virgin olive oil and refined soybean oil samples were analyzed by direct infusion, the identification of a natural lipid marker present only in soybean oil (m/z 886.68 [TAG+NH4]+) was possible. The certified extra virgin olive oil was purposely adulterated with soybean oil in different levels (1, 5, 10, 20, 50, 70, 90%) being possible to observe that the new methodology is able to detect even small fraud concentration, such as 1% (v/v). Additionally, commercial samples were analyzed and were observed the addition of soybean oil as a common fraud in this segment. This powerful analytical method proposed could be applied as routine analysis by control organization, as well as food industries, considering its pronounced advantages; simplicity, rapidity, elevated detectability and minor amounts of sample and solvent consumed.

LC-MS/MS analysis and evaluation of the anti-inflammatory activity of components from BushenHuoxue decoction.

CONTEXT: BushenHuoxue decoction (BSHXD) is a Chinese medicine prescription, which is composed of nine Chinese medical materials, used to treat osteoarthritis (OA). OBJECTIVE: This study develops sensitive and convenient LC-MS/MS methods to analyze chemical components from BSHXD, and assess the anti-inflammatory activities thereof. MATERIALS AND METHODS: The chemical composition from BSHXD water extract was qualitative analyzed by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS). Twelve reference compounds were analyzed by UPLC-ESI-MS/MS. Anti-inflammatory activities of target components were assessed by ELISA at 20 and 100 µg/mL. RESULTS: It is the first time that 88 compounds were qualitatively identified from BSHXD, of which 12 with potential in treating OA according to the literature were quantified. Within BSHXD the contents of quercetin, isopsoralen, icarisideII, osthole, and isoimperatorin increased remarkably compared with those in single herb which make up BSHXD, the contents were 0.1999, 0.4634, 0.0928, 0.5364, and 0.1487 mg/g. ELISA data displayed that BSHXD and the five compounds mentioned inhibited the expressions of TNF-α, IL-6 and NO released from LPS-stimulated RAW264.7 cell, with maximum inhibition rates of 104.05% (osthole, 100 µg/mL), 100.03% (osthole, 100 µg/mL), and 93.46% (isopsoralen, 20 µg/mL), respectively. DISCUSSION AND CONCLUSION: Content changes of 12 compounds in BSHXD and single herbs which comprise the prescription were measured and analyzed. Contents of five compounds increased may be explained by solubilization between drugs and chemical reaction. ELISA results reported that the increased contents of the five compounds could inhibit expression of the inflammatory factors.

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

Development of a Postcolumn Infused-Internal Standard Liquid Chromatography Mass Spectrometry Method for Quantitative Metabolomics Studies.

Quantitative metabolomics has become much more important in clinical research in recent years. Individual differences in matrix effects (MEs) and the injection order effect are two major factors that reduce the quantification accuracy in liquid chromatography-electrospray ionization-mass spectrometry-based (LC-ESI-MS) metabolomics studies. This study proposed a postcolumn infused-internal standard (PCI-IS) combined with a matrix normalization factor (MNF) strategy to improve the analytical accuracy of quantitative metabolomics. The PCI-IS combined with the MNF method was applied for a targeted metabolomics study of amino acids (AAs). D8-Phenylalanine was used as the PCI-IS, and it was postcolumn-infused into the ESI interface for calibration purposes. The MNF was used to bridge the AA response in a standard solution with the plasma samples. The MEs caused signal changes that were corrected by dividing the AA signal intensities by the PCI-IS intensities after adjustment with the MNF. After the method validation, we evaluated the method applicability for breast cancer research using 100 plasma samples. The quantification results revealed that the 11 tested AAs exhibit an accuracy between 88.2 and 110.7%. The principal component analysis score plot revealed that the injection order effect can be successfully removed, and most of the within-group variation of the tested AAs decreased after the PCI-IS correction. Finally, targeted metabolomics studies on the AAs showed that tryptophan was expressed more in malignant patients than in the benign group. We anticipate that a similar approach can be applied to other endogenous metabolites to facilitate quantitative metabolomics studies.

Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags.

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.

Development and validation of a liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of urolithin C in rat plasma and its application to a pharmacokinetic study.

Urolithins are microflora human metabolites of dietary ellagic acid derivatives. There is now a growing interest in the biological activities of these compounds. Several studies suggest that urolithins have potential antioxidant, anti-inflammatory, anticancer and anti-glycative activities. Recently, our group investigated the role of urolithins as potential anti-diabetic treatments; among the four urolithins, urolithin C was the most promising compound. The purpose of this paper was to develop a rapid, sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urolithin C in rat plasma. To date, no method is reported for the quantification of urolithin C in any of the matrices. Plasma samples were extracted with ethyl acetate. Urolithin D was selected as the internal standard. The separation was carried out on a C18 Kinetex EVO column (2.1mm×150mm, 2.6µm) using a mobile phase of acetonitrile-1% aqueous formic acid solution (30:70, v/v). A triple quadrupole mass spectrometer in the negative ion mode was used for the determination of the target analyte. The monitored ion transitions were m/z 243→187 for urolithin C and m/z 259→213 for the internal standard. The calibration curve range was 4.95-1085µg/L (r2>0.994). The intra- and inter-day precisions were less than 10%; accuracies ranged from 96.6 to 109%. The mean extraction recovery of urolithins C and D was greater than 91%. No significant matrix effects and no carryover effects were observed. Small changes in LC-ESI-MS/MS conditions did not have significant effect on the determination of urolithin C. Stability tests under various conditions were also investigated. This highly specific and sensitive method was used to analyze samples collected during preclinical pharmacokinetic studies in rats. Glucuronyl and sulfate conjugates of urolithin C were the main metabolites detected in plasma.

Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry.

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.

Determination of parabens in serum by liquid chromatography-tandem mass spectrometry: Correlation with lipstick use.

Parabens are the most widely used preservative and are considered to be relatively safe compounds. However, studies have demonstrated that they may have estrogenic activity, and there is ongoing debate regarding the safety and potential cancer risk of using products containing these compounds. In the present work, liquid chromatography-tandem mass spectrometry was applied to determine methylparaben and propylparaben concentrations in serum, and the results were correlated with lipstick application. Samples were analyzed using liquid-liquid extraction, followed by liquid chromatography-tandem mass spectrometry. The validation results demonstrated the linearity of the method over a range of 1-20 ng/mL, in addition to the method's precision and accuracy. A statistically significant difference was demonstrated between serum parabens in women who used lipstick containing these substances compared with those not using this cosmetic (p = 0.0005 and 0.0016, respectively), and a strong association was observed between serum parabens and lipstick use (Spearman correlation = 0.7202).

Rapid Identification and Assignation of the Active Ingredients in Fufang Banbianlian Injection Using HPLC-DAD-ESI-IT-TOF-MS.

Fufang Banbianlian Injection (FBI) is a well-known traditional Chinese medicine formula composed of three herbal medicines. However, the systematic investigation on its chemical components has not been reported yet. In this study, a high-performance liquid chromatography combined with diode-array detector, and coupled to an electrospray ionization with ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method, was established for the identification of chemical profile in FBI. Sixty-six major constituents (14 phenolic acids, 14 iridoids, 20 flavonoids, 2 benzylideneacetone compounds, 3 phenylethanoid glycosides, 1 coumarin, 1 lignan, 3 nucleosides, 1 amino acids, 1 monosaccharides, 2 oligosaccharides, 3 alduronic acids and citric acid) were identified or tentatively characterized by comparing their retention times and MS spectra with those of standards or literature data. Finally, all constituents were further assigned in the individual herbs (InHs), although some of them were from multiple InHs. As a result, 11 compounds were from Lobelia chinensis Lour, 33 compounds were from Scutellaria barbata D. Don and 38 compounds were from Hedyotis diffusa Willd. In conclusion, the developed HPLC-DAD-ESI-IT-TOF-MS method is a rapid and efficient technique for analysis of FBI sample, and could be a valuable method for the further study on the quality control of the FBI.