Online preconcentration of recombinant Arg-Gly-Asp-hirudin using dynamic pH junction for analysis in human urine samples by capillary electrophoresis-mass spectrometry

A sensitive and effort-saving method was established and validated for the quantitative determination of recombinant Arg-Gly-Asp-hirudin (rRGD-hirudin) in human urine samples. The assay was performed on a uncoated fused silica capillary of 70 cm × 50 μm I.D. and a positive voltage of 30 kV was applied. The sample was injected under pressure of 50 mbar for 300 s and the temperature of capillary was kept 25 °C. Sheath liquid consisting of 30% methanol and 70% of 0.1% formic acid aqueous solution flowing at 7 μL/min was supplied to the CE-electrospray interface. Utilizing the dynamic pH junction technique, a lower limit of quantitation of approximately 35 nM was achieved (concentration coefficiency was about 100-fold) without complex sample preprocessing procedure. CE-MS conditions and parameters were also optimized to obtain better performance. The method has been successfully applied in clinical research of rRGD-hirudin.     

CE‐ESI‐TOF/MS for human growth hormone analysis

CE is a powerful analytical tool used to separate intact biomolecules such as proteins. The coupling of CE with TOF/MS produces a very promising method that can be used to detect and identify proteins in different matrices. This paper describes an efficient, rapid, and simple CE‐ESI‐TOF/MS procedure for the analysis of endogenous human growth hormone and recombinant human growth hormone without sample preparation. Operational factors were optimized using an experimental design, and the method was successfully applied to distinguish human growth hormone and recombinant human growth hormone in unknown samples.     

Structural analysis of glycosphingolipid analogues obtained by the saccharide primer method using CE‐ESI‐MS

A glycosphingolipid analogue (12‐azidododecyl β‐lactoside) as a saccharide primer has been shown to be useful for the synthesis of oligosaccharide libraries by mammalian cells. In the present study, CE‐ESI‐MS was employed to elucidate the structure of glycosphingolipid analogues derived from 12‐azidododecyl β‐lactoside (Lac‐C12N3) by mammalian cells. MDCK cells and COLO201 cells were cultured with Lac‐C12N3, and the glycosylated products secreted into the medium were collected and separated into acidic and neutral products by column chromatography. The acidic products could be directly analyzed by CE‐ESI‐MS, while the neutral products were converted to anionic derivatives via a reaction with propiolic acid. With this method, it was possible to analyze both acidic and neutral products glycosylated by MDCK cells and COLO201 cells at high sensitivity.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

Quantification of 5‐aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5‐aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused‐silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R² = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50‐fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Doping control analysis of desmopressin in human urine by LC‐ESI‐MS/MS after urine delipidation

The World Anti‐Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high‐performance liquid chromatography–electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di‐isopropyl ether/n‐butanol and solid‐phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Quantification of the bisphosphonate alendronate using capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8–2000 ng/mL and an LOD of 2 ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8 min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.     

Dispersive solid‐phase extraction procedure coupled to UPLC‐ESI‐MS/MS analysis for the simultaneous determination of thirteen cytotoxic drugs in human urine

A fast and easy tailored dispersive solid‐phase extraction (d‐SPE) procedure has been developed for the determination of 13 cytostatic drugs. Combined with a rapid and simultaneous ultra performance liquid chromatography/tandem mass spectrometry method for residue identification and quantification in urine, it has been fully validated and tested to study a realistic situation in working environment. The target compounds were chosen from the most common classes used in hospitals. The d‐SPE adsorbent was obtained mixing Oasis HLB® with C₁₈ and applied to a large volume of sample (10 mL). The electrospray ionization‐mass spectrometry acquisition was conducted in a mixed period mode: six acquisition windows were in positive ionization and one in negative (for 5‐fluorouracil). The lowest limit of quantification was found at 0.04 μg/L urine for methotrexate. The absolute recovery of cytotoxic drugs was assessed at two concentrations levels and ranged from 67.1% (cytarabine) to 102.3% (etoposide) and from 65.3% (cytarabine) to 101.2% (methotrexate) for the lower and higher levels, respectively, with the relative standard deviation always <12%. This method gives the opportunity to analyze drugs in a wide molecular weight range (from 130 to 853 a.m.u.) and in a complex matrix, such as urine, without losing any of the features that a method intended for trace quantification must have. Copyright © 2016 John Wiley & Sons, Ltd.     

A simple fabrication method for tapered capillary tip and its applications in high‐speed CE and ESI‐MS

Fabrication of capillaries with tapered tips is an important technique that is required in many analytical chemistry areas, such as ESI‐MS, CE, electrochemical analysis, and microinjection. This paper describes a simple and effective grinding‐based fabrication method for capillaries with tapered tips. A novel grinding mode utilizing the combination of rotation and precession of an elastic capillary was developed, which significantly improved the controllability to the grinding process as well as the capillary tip shape. The capillary was fabricated by fixing it in an electric drill installed perpendicularly, and grind the capillary tip rotated around its own axis as well as the drill axis on sandpapers. Compared with conventional fabrication techniques for capillary tips, the present method is easy to control the capillary tip shape in routine laboratories without the requirement of expensive equipments or poisonous reagent (e.g. hydrofluoric acid (HF) solution). Various capillaries with different tip diameters and tip taper angles could be fabricated using the present method with good controllability and reproducibility. These capillaries were applied in high‐speed CE and ESI‐MS analysis to demonstrate the feasibility and potential of this fabrication method.     

Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti‐allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method was developed to screen, analyze and identify the anti‐allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC‐ESI‐MS/MS. The coupled system was then used to screen anti‐allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose‐dependent manner from 1 to 100 μm . The LAD2 cell membrane chromatography online with UHPLC‐ESI‐MS/MS method is an effective technique for screening the anti‐allergic components of Huangqi.     

Combination of dynamic pH junction with capillary electrophoresis‐mass spectrometry for the determination of systemins in plant samples

Systemin is an important group of plant peptide hormones participating in the regulation of plant defensive responses. An improved method, based on dynamic pH junction and capillary electrophoresis‐quadrupole time‐of‐flight mass spectrometry, was developed for online enrichment and sensitive determination of trace systemins in plants. After optimization, the online enrichment factors for six target systemins ranged from 90‐ to 127‐fold. The detection limits reached lower than 0.5 nM, which were comparable with the sensitivity of LC‐MS method. Satisfactory quantitative results were obtained in terms of linearity (R² ≥ 0.993), dynamic range (3–120 ng/mL), and reproducibility (≤6.7%). For the analysis of real plant samples, a rapid sample preparation method was developed, using two steps of SPE purification with different retention and separation mechanisms. Finally, this method realized the successful detection of tomato systemin and tobacco hydroxyproline‐rich systemin I from plant leaves with shorter analysis time.     

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10–100 folds. The dynamic pH junction method can be easily coupled with ESI‐MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200–400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.     

ESI‐MS Studies on Alcoholysis Mechanism of Electron‐deficient Cyclopropane Derivatives

Electrospray ionization mass spectrometry (ESI‐MS) was utilized to perform monitoring of the intermediates in the reaction of 1,2,3‐trisubstituted electron‐deficient cyclopropane derivatives, cis‐1‐thien‐2′‐oyl‐2‐(p‐subustituted phenyl‐6,6‐dimethyl)‐5,7‐dioxaspiro[2.5]‐4,8‐octadiones, with methanol. Key intermediates, either cationic or protonated forms of neutral species, were intercepted and characterized by ESI‐MS and its tandem version (ESI‐MS/MS). Therefore, the mechanism of the ring‐opening process for electron‐deficient cyclopropane derivatives was fully confirmed by the intermediates monitored.     

Simultaneous quantitative and qualitative analysis of aliskiren,enalapril and its active metabolite enalaprilat in undiluted human urine utilizing LC‐ESI‐MS/MS

The benefit–risk ratio of combined blocking by the direct renin inhibitor aliskiren and an angiotensin‐converting enzyme inhibitor (e.g. enalapril) on the renin–angiotensin–aldosterone system is discussed. No method was available for simultaneous determination of both drugs in urine. A novel sensitive method for simultaneous quantification in undiluted human urine was developed which enables systematic pharmacokinetic investigations, especially in poorly investigated populations like children. Matrix effects were clearly reduced by applying solid‐phase extraction followed by a chromatographic separation on Xselect^TM C₁₈ CSH columns. Mobile phase consisted of methanol and water, both acidified with formic acid. Under gradient conditions and a flow rate of 0.4 mL/min the column effluent was monitored by tandem mass spectrometry with electrospray ionization. Calibration curves were constructed in the range of 9.4–9600 ng/mL regarding aliskiren, 11.6–12000 ng/mL for enalapril and 8.8–9000 ng/mL for enalaprilat. All curves were analyzed utilizing 1/x²‐weighted quadratic squared regression. Intra‐run and inter‐run precision were 3.2–5.8% and 6.1–10.3% for aliskiren, 2.4–6.1% and 3.9–7.9% for enalapril as well as 3.1–9.4% and 4.7–12.7% regarding enalaprilat. Selectivity, accuracy and stability results comply with current international bioanalysis guidelines. The fully validated method was successfully applied to a pharmacokinetic investigation in healthy volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI‐MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI‐MS. Using EKS, the sensitivity of the method was improved 5000‐fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE‐ESI‐MS/MS to analyze biological samples.     

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC‐ESI‐MS/MS

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole derivatives and liquid chromatography–electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N¹‐acetylputrescine (N¹‐actPUT), N⁸‐acetylspermidine, N¹‐acetylspermine, 1,3‐diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal‐to‐noise ratio 5) were 5–100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6‐diaminohexane, vs the injected amounts of polyamines (r² > 0.996), and the intra‐day and inter‐day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14–110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N¹‐actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of sildenafil and N‐desmethyl sildenafil in human plasma by UFLC coupled with ESI‐MS/MS and pharmacokinetic and bioequivalence studies in Malay population

A simple, rapid, specific and reliable UFLC coupled with ESI‐MSMS assay method to simultaneously quantify sildenafil and N‐desmethyl sildenafil, with loperamide as internal standard, was developed. Chromatographic separation was performed on a Thermo Scientific Accucore C₁₈ column with an isocratic mobile phase composed of 0.1% v/v formic acid in purified water–methanol (20:80, v/v), at a flow rate of 0.3 mL/min. Sildenafil, N‐desmethyl sildenafil and loperamide were detected with proton adducts at m/z 475.4 > 58.2, 461.3 > 85.2 and 477.0 > 266.1 in multiple reaction monitoring positive mode, respectively. Both analytes and internal standard were extracted by diethyl ether. The method was validated over a linear concentration range of 10–800 ng/mL for sildenafil and 10–600 ng/mL for N‐desmethyl sildenafil with correlation coefficient (r²) ≥0.9976 for sildenafil and (r²) ≥0.9992 for N‐desmethyl sildenafil. The method was precise, accurate and stable. The proposed method was applied to study the bioequivalence between a 100 mg dose of two pharmaceutical products: Viagra (original) and Edyfil (generic) products. AUC_0–t, C_max and T_max were 2285.79 ng h/mL, 726.10 ng/mL and 0.94 h for Viagra and 2363.25 ng h/mL, 713.91 ng/mL and 0.83 hour for Edyfil. The 90% confidence interval of these parameters of this study fall within the regulatory range of 80–125%, hence they are considered as bioequivalent. Copyright © 2014 John Wiley & Sons, Ltd.