Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.     

Astragalus polysaccharide improves palmitate-induced insulin resistance by inhibiting PTP1B and NF-κB in C2C12 myotubes

We investigated the effects of Astragalus polysaccharide (APS) on palmitate-induced insulin resistance in C2C12 skeletal muscle myotubes. Palmitate-reduced glucose uptake was restored by APS. APS prevented palmitate-induced C2C12 myotubes from impaired insulin signaling by inhibiting Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and increasing Ser473 phosphorylation of Akt. Moreover, the increases in protein-tyrosine phosphatase-1B (PTP1B) protein level and NF-κB activation associated with palmitate treatment were also prevented by APS. However the treatment with APS didn't change AMP-activated protein kinase (AMPK) activation in palmitate-induced myotubes. The results of the present study suggest that Astragalus polysaccharide inhibits palmitate-induced insulin resistance in C2C12 myotubes by inhibiting expression of PTP1B and regulating NF-κB but not AMPK pathway.     

Label-Free Proteomic Identification of Endogenous,Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

Protein–protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein–protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein–protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.     

LC-MS Quantification of Site-Specific Phosphorylation Degree by Stable-Isotope Dimethyl Labeling Coupled with Phosphatase Dephosphorylation

Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO₂ enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.     

Observation of higher order quadrupole excitation frequencies in a linear ion trap

The resonant frequencies for quadrupole excitation of ions confined with a buffer gas in a linear quadrupole ion trap with Mathieu parameters a = 0 and q ∼ 0.36 have been measured. The resonances are predicted to occur at angular frequencies ω _n ^K given by ω _n ^K = (Ώ/K)|n + β| without the presence of a buffer gas where ϒ is the angular frequency of the trapping radio frequency, K = 1,2,3 … is the order of the resonance calculated with perturbation theory, and n = 0, ±1, ±2, ±3 …. The resonances are measured through fragmentation of protonated reserpine. The observed frequencies agree closely with the theoretical values but there are small differences which vary from +0.6% at K = 2 to −2.7% at K = 6. This is believed to be the result of the dependence of the resonant frequencies upon the buffer gas density and/or the excitation amplitude. The resolution of the resonances (measured from the depletion of precursor and formation of fragment ions) increased by a factor of 2 as K increased from 1 to 6. This increase in resolution warrants further investigation into the use of higher order resonances for isolation and excitation of trapped ions.     

Mapping <Emphasis Type="Italic">N</Emphasis>-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a₁ signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a₁ ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a₁ ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H₃PO₄ ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a₁ ion enhancement due to the resulting unsaturated covalent bond on C _α of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.     

Copolymerization of glycidyl methacrylate with alkyl acrylate monomers

A newer approach to obtaining acrylic thermoset polymers with adequate hydrophilicity required for various specific end uses is reported. Glycidyl methacrylate (GMA) was copolymerized with n-butyl acrylate (n-BA), isobutyl acrylate (i-BA), and 2-ethylhexyl acrylate (2-EHA) in bulk at 60°C. with benzoyl peroxide as free radical initiator. The copolymer composition was determined from the estimation of epoxy group. Reactivity ratios were calculated by the Yezrielev, Brokhina, and Roskin method. For copolymerization of GMA (M₁) with n-BA (M₂) the reactivity ratios were r₁ = 2.15 ± 0.14, r₂ = 0.12 ± 0.03; with i-BA (M₂) they were r₁ = 1.27 ± 0.06, r₂ = 0.33 ± 0.031; and with 2-EHA (M₂) they were r₁ = 2.32 ± 0.14, r₂ = 0.13 ± 0.009. The reactivity ratios were the measure of distribution of monomer units in a copolymer chain; the values obtained are compared and discussed.     

质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、 α1-抗胰蛋白酶(A1AT)、 α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、 位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究. 利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子, 用Byonic软件发现了新的糖基化位点信息, 即增加了原位点处聚糖糖型的种类. 对于A2HSG, 新增了N-糖基化156位点上的4种糖型, N-糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和O-糖基化346位点上的1种糖型. 对于Apo C3, 只有O-糖基化94一个位点, 在此位点上新增了9种糖型. 同时, 调整了用于定量蛋白的多肽, 使得定量更加准确. 采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为, 并重新计算了每种聚糖的位点占有率, 优化了现有的定量方法.     

A laser flash photolysis/IR diode laser absorption study of the reaction of chlorine atoms with selected alkanes

A high‐resolution IR diode laser in conjunction with a Herriot multiple reflection flow‐cell has been used to directly determine the rate coefficients for simple alkanes with Cl atoms at room temperature (298 K). The following results were obtained: k(Cl + n‐butane) = (1.91 ± 0.10) × 10^?10 cm³ molecule^?1 s^?1, k(Cl + n‐pentane) = (2.46 ± 0.12) × 10^?10 cm³ molecule^?1 s^?1, k(Cl + iso‐pentane) = (1.94 ± 0.10) × 10^?10 cm³ molecule^?1 s^?1, k(Cl + neopentane) = (1.01 ± 0.05) × 10^?10 cm³ molecule^?1 s^?1, k(Cl + n‐hexane) = (3.44 ± 0.17) × 10^?10 cm³ molecule^?1 s^?1 where the error limits are ±1σ. These values have been used in conjunction with our own previous measurements on Cl + ethane and literature values on Cl + propane and Cl + iso‐butane to generate a structure activity relationship (SAR) for Cl atom abstraction reactions based on direct measurements. The resulting best fit parameters are k_p = (2.61 ± 0.12) × 10^?11 cm³ molecule^?1 s^?1, k_s = (8.40 ± 0.60) × 10^?11 cm³ molecule^?1 s^?1, k_t = (5.90 ± 0.30) × 10^?11 cm³ molecule^?1 s^?1, with f( ? CH₂^? ) = f (? CH₂^? ) = f (?C?) = f = 0.85 ± 0.06. Tests were carried out to investigate the potential interference from production of excited state HCl(v = 1) in the Cl + alkane reactions. There is some evidence for HCl(v = 1) production in the reaction of Cl with shape n‐hexane. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 34: 86–94, 2002     

Laser desorption ionization of red phosphorus clusters and their use for mass calibration in time‐of‐flight mass spectrometry

Phosphorus clusters P_n (n = 1–89) are easily formed from red phosphorus by laser desorption ionization (LDI) and they cover a range of up to approx. m/z 3000 in both positive and negative ion mode. The clusters are singly charged and the spectra are simple because phosphorus is monoisotopic. The mass spectra can be measured with an acceptable resolution and intensity. The use of positively charged P_n clusters for calibration in mass spectrometry was examined and it was demonstrated that in external calibration a standard deviation of ±0.04 m/z units can be achieved even when using a common commercial matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) instrument. When used as internal standards the P_n clusters react with some analytes – C₆₀ and C₇₀ fullerenes and cucurbituril[8], for example. It was also found that red phosphorus is a suitable MALDI matrix for peptides and proteins, illustrated by the examples of a Calmix mixture of bradykinin, angiotensin, renin, adrenocorticotropic hormone ACTH fragment 18‐359 and insulin, and of insulin alone. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and quantification of atractylenolide I and atractylenolide III in Rhizoma Atractylodes Macrocephala by liquid chromatography–ion trap mass spectrometry

Rhizoma Atractylodes Macrocephala (RAM) is an important traditional Chinese medicinal herb that is used for treatment of dyspepsia and anorexia. The active ingredients, atractylenolide I (AO‐I) and atractylenolide III (AO‐III), were identified by direct‐injection ion trap‐mass spectrometry (IT‐MS) for collecting MSⁿ spectra. The major fragment ions of AO‐I and AO‐III were confirmed by MSⁿ both in negative ion mode and in positive ion mode. The possible main cleavage pathway of fragment ions was studied. The determinations of AO‐I and AO‐III were accomplished by liquid chromatography (LC) with UV and MS. The analytes provided good signals corresponding to the protonated molecular ions [M + H]⁺ and product ions. The precursor ions and product ions for quantification of AO‐III and AO‐I were m/z 249 → 231 and m/z 233 → 215, respectively, using selected ion monitoring by LC‐IT‐MS. Two methods were evaluated for a number of validation characteristics (repeatability, limit of detection, calibration range, and recovery). MS provides a high selectivity and sensitivity for determination of AO‐III and AO‐I in positive mode. After optimization of the methods, separation, identification and quantification of the two components in RAM were comprehensively tested by HPLC with UV and MS. Copyright © 2012 John Wiley & Sons, Ltd.     

A reversed-phase ultra-fast liquid chromatography–photodiode array detector (RP-UFLC-PDA) method for simultaneous estimation of ayapanin and umbelliferone in Ayapana triplinervis Vahl

A rapid validated ultra-fast liquid chromatography–photodiode array detector (UFLC-PDA) method was developed to identify and quantify ayapanin (AY) and umbelliferone (UM) simultaneously in Ayapana triplinervis Vahl methanolic extract. The method was validated for linearity, limit of detection (LOD; 3:1σ/S), limit of quantification (LOQ; 10:1σ/S), precision, accuracy, specificity and robustness. The response was linear with a good correlation between concentration and mean peak area through a correlation coefficient of 0.9996, y = 7025.7x – 2269.8 and 0.9997, y = y = 16,262x – 946 with LOD of 6.256 ± 0.52 and 3.325 ± 0.36, and LOQ of 18.838 ± 0.18 and 8.870 ± 0.85 for AY (0.67% w/w) and UM (0.18% w/w), respectively. The relative standard deviation (%) of precision and recovery of AY and UM was <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.     

Quantification of methyllycaconitine,selective α7 nicotinic receptor antagonist,in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry – application to neuropharmacokinetics of methyllycaconitine in rats

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 683–216 for MLA and m/z 260–116 for the internal standard. The assay exhibited a linear dynamic range of 0.5–250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study. Copyright © 2011 John Wiley & Sons, Ltd.     

Rate constants for the gas-phase reactions of Cl-atoms with C2?C8 alkanes at T = 296 ± 2 K

Relative rate constants for the gas-phase reactions of Cl-atom with thirteen atmospherically interesting alkanes (C₂? C₈) have been determined at 296 ± 2 K based on GC/FID measurements of their relative decays in the UV (λ ≥ 300 nm) photolysis of mixtures containing Cl₂ and the entire series of the selected alkanes in the mtorr range in 750 torr of N₂. The following absolute rate constants (in units of 10^?10 cm³ molecule^?1 s^?1) have been derived from the relative rate constants combined with the value of 1.94 × 10^?10 cm³ molecule^?1 s^?1 for the Cl + n-butane reaction: ethane (0.57 ± 0.05); propane (1.27 ± 0.02); 2-methyl propane (1.30 ± 0.01), 2-methyl butane ((1.96 ± 0.02)), n-pentane (2.50 ± 0.02); 2,3-dimethyl butane (2.00 ± 0.06); 2-methyl pentane (2.58 ± 0.08); n-hexane (3.05 ± 0.04); 2-methyl hexane (3.12 ± 0.04); n-heptane (3.65 ± 0.06); 2,2,4-trimethyl pentane (2.25 ± 0.08); and n-octane (4.09 ± 0.12). The uncertainties indicated are two least-squares standard deviations (2σ). These rate constants are compared with literature values and their applicability to Arctic tropospheric conditions is discussed. © 1995 John Wiley & Sons, Inc.     

Temperature dependence of the rate constants of the reactions of oxygen atoms with 1-pentene, 1-hexene,cis-2-pentene,and trans-2-pentene

The kinetics of the reactions of ground state oxygen atoms with 1-pentene, 1-hexene, cis-2-pentene, and trans-2-pentene was investigated in the temperature range 200 to 370 K. In this range the temperature dependences of the rate constants can be represented by k = A′ Tⁿ exp(− E′_a/RT) with A′ = (1.0 ± 0.6) · 10⁻¹⁴ cm³ s⁻¹, n = 1.13 ± 0.02, E′_a = 0.54 ± 0.05 kJ mol⁻¹ for 1-pentene: A′ = (1.3 ± 1.2) · 10⁻¹⁴ cm³ s⁻¹, n = 1.04 ± 0.08, E′_a = 0.2 ± 0.4 kJ mol⁻¹ for 1-hexene; A′ = (0.6 ± 0.6) · 10⁻¹⁴ cm³ s⁻¹, n = 1.12 ± 0.05, E′_a = − 3.8 ± 0.8 kJ mol⁻¹ for cis-2-pentene; and A′ = (0.6 ± 0.8) · 10⁻¹⁴ cm³ s⁻¹, n = 1.14 ± 0.06, E′_a = − 4.3 ± 0.5 kJ mol⁻¹ for trans-2-pentene. The atoms were generated by the H₂-laser photolysis of NO and detected by time resolved chemiluminescence in the presence of NO. The concentrations of the O(³P) atoms were kept so low that secondary reactions with products are unimportant. © 1997 John Wiley & Sons, Inc.     

Formation of positive cluster ions LinBr (n = 2–7) and ionization energies studied by thermal ionization mass spectrometry

Clusters of the type Li_nX (X = halides) can be considered as potential building blocks of cluster‐assembly materials. In this work, Li_nBr (n = 2–7) clusters were obtained by a thermal ionization source of modified design and selected by a magnetic sector mass spectrometer. Positive ions of the Li_nBr (n = 4–7) cluster were detected for the first time. The order of ion intensities was Li₂Br⁺ > Li₄Br⁺ > Li₅Br⁺ > Li₆Br⁺ > Li₃Br⁺. The ionization energies (IEs) were measured and found to be 3.95 ± 0.20 eV for Li₂Br, 3.92 ± 0.20 eV for Li₃Br, 3.93 ± 0.20 eV for Li₄Br, 4.08 ± 0.20 eV for Li₅Br, 4.14 ± 0.20 eV for Li₆Br and 4.19 ± 0.20 eV for Li₇Br. All of these clusters have a much lower ionization potential than that of the lithium atom, so they belong to the superalkali class. The IEs of Li_nBr (n = 2–4) are slightly lower than those in the corresponding small Li_n or Li_nH clusters, whereas the IEs of Li_nBr are very similar to those of Li_n or Li_nH for n = 5 and 6. The thermal ionization source of modified design is an important means for simultaneously obtaining and measuring the IEs of Li_nBr (n = 2–7) clusters (because their ions are thermodynamically stable with respect to the loss of lithium atoms in the gas phase) and increasingly contributes toward the development of clusters for practical applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1–500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C_max; 616 ± 73 ng/mL) was achieved at 0.5 h (T_max) and area under curve (AUC_0–24) was 1841 ± 308 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials. Copyright © 2011 John Wiley & Sons, Ltd.     

An integrated strategy for identification and relative quantification of site-specific protein phosphorylation using liquid chromatography coupled to MS2/MS3

Reversible and differential multisite protein phosphorylation is an important mechanism controlling the activity of cellular proteins. Here we describe a robust and highly selective approach for the identification and relative quantification of site-specific phosphorylation events. This integrated strategy has three major parts: visualisation of phosphorylated proteins using fluorescently stained polyacrylamide gels, determination of the phosphorylation site(s) using automatic MS3 triggered by the loss of phosphoric acid, and relative quantification of phosphorylation by integrating MS2- and MS3-extracted ion traces using a fast-scanning, linear ion trap mass spectrometer. As a test case, recombinant sucrose-phosphate synthase (SPS) from Arabidopsis thaliana (At5g1110) was used for identification and quantification of site-specific phosphorylation. The identified phosphorylation site of the actively expressed protein coincides with the major regulatory in vivo phosphorylation site in spinach SPS. Site-specific differential in vitro phosphorylation of native protein was demonstrated after incubation of the recombinant protein with cold-adapted plant leaf extracts from A. thaliana, suggesting regulatory phosphorylation events of this key enzyme under stress response.     

Biological activity and LC-MS profiling of ethyl acetate extracts from Nitraria sibirica (Pall.) fruits

Nitraria sibirica is a traditional Uighur medicine. This study was undertaken to investigate the bioactivity of N. sibirica fruit extract and to evaluate their chemical compositions. The ethyl acetate extract from N. sibirica fruits exhibited the potential antioxidant activity (SC₅₀ = 30.17 ± 0.06 μg/mL) and protein tyrosine phosphatase 1B inhibitory activity (IC₅₀ = 7.15 ± 0.03 μg/mL) in vitro. In order to investigate the active constituents in this extract, a LC-QTOF-MS/MS method was developed and established. A total of 28 compounds including seven cinnamic acids, nine benzoic acids and 12 flavonoids were identified or partially characterised according to the accurate mass and the characteristic fragment ions at low and high collision energy. Most of them were reported for the first time in this plant. Phytochemical profiles of the active extract will help the development and utilisation of N. sibirica in food and medicine.     

Characterization and quantification of 10‐hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of 10‐hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MSⁿ spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI‐MSⁿ fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MSⁿ in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative‐ion mode at a product ion at m/z 363 → 319 by LC‐MS. The LC‐MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC‐MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time. Copyright © 2013 John Wiley & Sons, Ltd.