Taylor-Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins.

As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.

CZE-MS peptide mapping: To desalt or not to desalt?

BACKGROUND: In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices. RESULTS: First, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies. SIGNIFICANCE: Based on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.

Determination of human insulin and its six therapeutic analogues by capillary electrophoresis - mass spectrometry.

In this work, human insulin and its 6 analogues were separated and determined using CZE-MS. Three different capillaries (bare fused silica, successive multiple ionic-polymer layer (SMIL) and static linear polyacrylamide (LPA) coated) were compared based on their separation performances in their optimal operating conditions. Coated capillaries demonstrated slightly better separation of the components, although some components showed wide, distorted peaks. The highest plate number could be obtained in the SMIL capillary (192 000/m). For UV and ESI-MS detection relatively similar LOD values were obtained (0.3-1.2 mg/L and 1.0-3.4 mg/L, respectively). The application of MS detection provided useful structural information and unambiguous identification for insulins having similar or the same molecular mass. This work is considered to be important not only for the investigation of insulins but also for its potential contribution to the top-down analysis of proteins using CE-MS.

Determination of artemisinin and its analogs in Artemisia annua extracts by capillary electrophoresis - Mass spectrometry.

The applicability of micellar electrokinetic capillary chromatography (MEKC) with mass spectrometric detection for the determination of artemisinin and its analogs (e.g. ascaridole, artemisia ketone, casticin, deoxyartemisinin, arteannuic acid, artemetin, dihydroartemisinic acid) was studied. 40 mM ammonium perfluorooctanoate (pH 9.5) with 2% isopropanol (IPA) was used as background electrolyte (BGE) and the sheath liquid was 50 % (v/v) IPA:water containing 0.1 % formic acid. Separation was performed in a bare fused silica capillary. Artemisinin was detected at 283.1545 m/z as [M+H]+ ion. For artemisinin the linear range was found to be 0.6 µg/mL - 60 µg/mL and the limit of detection was 0.18 µg/mL. The RSD% values were 2.6 % for migration times and 4.8 % for peak areas (N = 6). In the ethanolic extracts of Artemisia annua leaves, in addition to artemisinin, a large number of other organic components could be separated and determined. MEKC-MS revealed the existence of diastereomers of several compounds (artemisinin, deoxyartemisinin, dihydroartemisinic acid) in the plant extracts.

High Enzyme Activity of a Binuclear Nickel Complex Formed with the Binding Loops of the NiSOD Enzyme*.

Detailed equilibrium, spectroscopic and superoxide dismutase (SOD) activity studies are reported on a nickel complex formed with a new metallopeptide bearing two nickel binding loops of NiSOD. The metallopeptide exhibits unique nickel binding ability and the binuclear complex is a major species with 2×(NH2 ,Namide ,S- ,S- ) donor set even in an equimolar solution of the metal ion and the ligand. Nickel(III) species were generated by oxidizing the NiII complexes with KO2 and the coordination modes were identified by EPR spectroscopy. The binuclear complex formed with the binding motifs exhibits superior SOD activity, in this respect it is an excellent model of the native NiSOD enzyme. A detailed kinetic model is postulated that incorporates spontaneous decomposition of the superoxide ion, the dismutation cycle and fast redox degradation of the binuclear complex. The latter process leads to the elimination of the SOD activity. A unique feature of this system is that the NiIII form of the catalyst rapidly accumulates in the dismutation cycle and simultaneously the NiII form becomes a minor species.

Analysis of honey using capillary electrophoresis with electrokinetic injection - Direct analysis of viscous samples without sample pretreatment.

Although the electrokinetic injection mode is applicable for direct introduction of ionic components from highly viscous samples, this is the first work which studies this special feature of capillary electrophoresis for this purpose. 9 metal cations were determined in honey utilizing direct injection, zone electrophoretic separation and indirect UV detection of the sample components. Reproducibility better than 4 RSD% was found for peak areas and the LOD values ranged between 0.045 and 0.44 mg/kg when injection parameters were set to 7.5 kV × 5 s. In order to remedy the quantitation problems arising from mobility and matrix bias, the applicability of the internal universal calibration was examined. It was found that in cases where the mobilities of cations do not differ from the mobilities of the monitoring ion and the internal universal standard (IUS) by more than 10%, the obtained results agreed well with the data measured with ICP-OES.

Chemometric Assessment of Chromatographic Methods for Herbal Medicines Authentication and Fingerprinting.

Nowadays, increasingly more individuals turn to supplementation of the diet with herbal medicines and many such products are marketed lately. Thus the problem that this article focuses on is that these products are not subjected to rigorous quality control like synthetic drugs are, which rises a constant debate whether the supplements actually contain the herb or mixture of herbs that the manufacturer claims they do. As a solution, micellar electrokinetic chromatography and high performance liquid chromatography were investigated in order to fingerprint and authenticate herbal medicines. For this purpose, minimal sample pre-treatment was applied to several fruit based herbal medicines, which were compared with the ethanolic extract of the respective fruit. The holistic evaluation of the electropherograms and chromatograms was made by using appropriate chemometric tools, such as principal component analysis (PCA), cluster analysis and a combination of PCA and linear discriminant analysis (PCA-LDA). The results suggest that the developed method was able to successfully discriminate between different herbal medicines, based on their raw material content. Moreover, this simple and efficient methodology might also be used for routine screening and authenticity control of different products and could be implemented in any quality control laboratory.

The role of equilibrium and kinetic properties in the dissociation of Gd[DTPA-bis(methylamide)] (Omniscan) at near to physiological conditions.

[Gd(DTPA-BMA)] is the principal constituent of Omniscan, a magnetic resonance imaging (MRI) contrast agent. In body fluids, endogenous ions (Zn(2+), Cu(2+), and Ca(2+)) may displace the Gd(3+). To assess the extent of displacement at equilibrium, the stability constants of DTPA-BMA(3-) complexes of Gd(3+), Ca(2+), Zn(2+), and Cu(2+) have been determined at 37 °C in 0.15 M NaCl. The order of these stability constants is as follows: GdL≈CuL>ZnL≫CaL. Applying a simplified blood plasma model, the extent of dissociation of Omniscan (0.35 mM [Gd(DTPA-BMA)]) was found to be 17% by the formation of Gd(PO4), [Zn(DTPA-BMA)](-) (2.4%), [Cu(DTPA-BMA)](-) (0.2%), and [Ca(DTPA-BMA)](-) (17.7%). By capillary electrophoresis, the formation of [Ca(DTPA-BMA)](-) has been detected in human serum spiked with [Gd(DTPA-BMA)] (2.0 mM) at pH 7.4. Transmetallation reactions between [Gd(DTPA-BMA)] and Cu(2+) at 37 °C in the presence of citrate, phosphate, and bicarbonate ions occur by dissociation of the complex assisted by the endogenous ligands. At physiological concentrations of citrate, phosphate, and bicarbonate ions, the half-life of dissociation of [Gd(DTPA-BMA)] was calculated to be 9.3 h at pH 7.4. Considering the rates of distribution and dissociation of [Gd(DTPA-BMA)] in the extracellular space of the body, an open two-compartment model has been developed, which allows prediction of the extent of dissociation of the Gd(III) complex in body fluids depending on the rate of elimination of the contrast agent.

A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M).

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 µg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 µL was analyzed. The obtained peak concentrations (8.2-10.1 µg/mL) and T(max) (44-65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 µg/g.

Determination of gadolinium-based magnetic resonance imaging contrast agents by micellar electrokinetic capillary chromatography.

MEKC with DAD was applied to detect six Gd-based contrasting agents (CAs) (Gd-DTPA-BMA (Omniscan), Gd-HPDO3A (ProHance), Gd-DOTA (Dotarem), Gd-AAZTA, Gd-BOPTA (Multihance) and Gd-DTPA (Magnevist)) commonly used in MRI diagnostics. The achieved LODs ranged between 0.40 and 20 µM and the optimized method gave excellent precision, especially when two internal standards were applied (less than 0.34 RSD% for migration time). The MEKC technique made it possible to determine the CAs in urine and serum samples of patients having a therapeutic dose. Due to the SDS content of the running buffer, the serum samples can be directly injected to analyze Gd-based CAs without interference of high protein content.

Analysis and stability study of temozolomide using capillary electrophoresis.

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31-0.93 microg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.

Role of pathogenic oral flora in postoperative pneumonia following brain surgery.

BACKGROUND: Post-operative pulmonary infection often appears to result from aspiration of pathogens colonizing the oral cavity. It was hypothesized that impaired periodontal status and pathogenic oral bacteria significantly contribute to development of aspiration pneumonia following neurosurgical operations. Further, the prophylactic effects of a single dose preoperative cefazolin on the oral bacteria were investigated. METHODS: A matched cohort of 18 patients without postoperative lung complications was compared to 5 patients who developed pneumonia within 48 hours after brain surgery. Patients waiting for elective operation of a single brain tumor underwent dental examination and saliva collection before surgery. Bacteria from saliva cultures were isolated and periodontal disease was scored according to type and severity. Patients received 15 mg/kg cefazolin intravenously at the beginning of surgery. Serum, saliva and bronchial secretion were collected promptly after the operation. The minimal inhibitory concentrations of cefazolin regarding the isolated bacteria were determined. The actual antibiotic concentrations in serum, saliva and bronchial secretion were measured by capillary electrophoresis upon completion of surgery. Bacteria were isolated again from the sputum of postoperative pneumonia patients. RESULTS: The number and severity of coexisting periodontal diseases were significantly greater in patients with postoperative pneumonia in comparison to the control group (p = 0.031 and p = 0.002, respectively). The relative risk of developing postoperative pneumonia in high periodontal score patients was 3.5 greater than in patients who had low periodontal score (p < 0.0001). Cefazolin concentration in saliva and bronchial secretion remained below detectable levels in every patient. CONCLUSION: Presence of multiple periodontal diseases and pathogenic bacteria in the saliva are important predisposing factors of postoperative aspiration pneumonia in patients after brain surgery. The low penetration rate of cefazolin into the saliva indicates that its prophylactic administration may not be sufficient to prevent postoperative aspiration pneumonia. Our study suggests that dental examination may be warranted in order to identify patients at high risk of developing postoperative respiratory infections.

A comparative study of capillary zone electrophoresis and pH-potentiometry for determination of dissociation constants.

Acidity constants of six cephalosporin antibiotics, cefalexin, cefaclor, cefadroxil, cefotaxim, cefoperazon and cefoxitin are determined using capillary zone electrophoresis (CZE) and pH-potentiometric titrations. Since CZE is a separation method, it is not necessary for the samples to be of high purity and known concentration because only mobilities are measured. The effect on determination of dissociation constants of different matrices (serum, 0.9% NaCl, fermentation matrix) was examined. The advantages of CZE can be utilized in those fields where potentiometry has limitations (sample quantity, solubility, purity, simultaneous determinations), although pK(a) values that are close to each other can be determined by potentiometry with more accuracy.

Analysis of cephalosporins in bronchial secretions by capillary electrophoresis after simple pretreatment.

The applicability of capillary zone electrophoresis (CZE) for analysis of cephalosporin antibiotics has been studied in bronchial secretion as highly viscous, thick and non-homogeneous samples. The lyophilization was found to be a simple but effective pretreatment of these samples to bring them into a form which is suitable for injection to CE capillary. The obtained good recovery data prove that the lyophilization/dissolution of bronchial secretion samples can be reproducibly performed.

Effectiveness of cephalosporins in the sputum of patients with nosocomial bronchopneumonia.

Nosocomial bronchopneumonia is a frequent complication in patients with chronic intratracheal intubation. Despite targeted antibiotic treatment, production of abundant bronchial secretion containing pathogen bacteria often tends to be chronic, and so mortality drastically increases. This problem led to an investigation of the penetration of five cephalosporin antibiotics into the sputum. Serum and sputum were collected from 24 chronically intubated patients having purulent nosocomial bronchopneumonia treated in an intensive care unit (ICU). Patients received the following doses intravenously every 24 h: five received 70 mg/kg of body weight cefuroxime, four received 110 mg/kg cefamandole, six received 80 mg/kg ceftriaxone, four received 80 mg/kg ceftazidime, and five received 80 mg/kg cefepime. Antibiotic concentrations in the serum and sputum were evaluated by capillary electrophoresis. MICs were determined for bacteria isolated from the purulent bronchial secretions. The mean levels of the cephalosporins in the sputum did not reach the MICs for the bacteria isolated from the same samples. Ceftriaxone was the only one of the investigated five cephalosporins that had a measurable concentration in the sputum (1.4 +/- 1.2 mg/liter). The low concentration of antibiotics in the purulent tracheobronchial secretion can be one of the many reasons for ineffective therapy of nosocomial bronchopneumonia in intubated patients in the ICUs. In the case of intubated or mechanically ventilated patients having chronic bronchopneumonia, determination of drug concentration in the bronchial secretion might be considered when selecting an antibiotic for treatment.

Application of capillary zone electrophoresis to the analysis and to a stability study of cephalosporins.

The applicability of capillary zone electrophoresis (CZE) for the determination of cephalosporin antibiotics has been studied. In the case of the separation conditions optimised for fourteen cephalosporins, the precision of migration times was smaller than 1.3% RSD, and the values of the limit of detection ranged between 0.42 and 1.62 microg/ml. The proposed CZE method was applied to study the stability of cephalosporins in water at different temperatures (+25, +4 and -18 degrees C). It was established that the degradation of most cephalosporins was not higher than 20% at room temperature within 4 h of dissolution of these antibiotics.