Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

Utility of membrane preconcentration-capillary electrophoresis-mass spectrometry in overcoming limited sample loading for analysis of biologically derived drug metabolites, peptides, and proteins

The limited loading of capillary electrophoresis (CE) leads to relatively poor concentration limits of detection. In this work a unique method for analyte preconcentration with capillary electrophoresis-mass spectrometry (CE-MS) is described. A cartridge containing an impregnated membrane is installed at the inlet of the CE capillary, and we term this approach membrane preconcentration capillary electrophoresis-mass spectrometry (mPC-CE-MS). The analysis of in vivo derived metabolites, peptides, and proteins is described showing the wide applicability of the technology in the analysis of numerous compound classes ranging in molecular weight from 200–60,000 u. In particular, we describe the direct mPC-CE-MS analysis of urine obtained from a patient receiving the neuroleptic drug haloperidol. Three metabolites were found in the urine, and two of them are implicated in the Parkinsonian-like side effects caused by taking this drug. The technique is also applied to the analysis of major histocompatibility complex class I peptides obtained from EG-7 cells. Furthermore, the clinical potential of this approach is described by the direct analysis of urine from a patient suffering from multiple myeloma, as well as aqueous humor derived from a patient undergoing surgery. Finally we show that the use of mPC-ME-MS in conjunction with either analyte stacking (small organic molecules such as metabolites) or moving-boundary transient isotachophoresis (peptides and proteins) after analytes have been eluted from the adsorptive membrane affords optimal performance and no compromise in CE mass spectrometry performance.     

Optimization of an isotope dilution gas chromatography/mass spectrometry method for the detection of endogenous estrogen metabolites in urine samples

A gas chromatography/mass spectrometry (GC/MS) method for the simultaneous quantitation of ten estrogen metabolites in human urine was optimized. The method consists of initial enzymatic hydrolysis of the estrogen conjugates using beta-glucuronidase followed by solid-phase extraction (SPE) on Sep-pak C18 columns and further sample purification by ion-exchange chromatography on QAE-Sephadex cartridges in the acetate form. QAE-Sephadex cartridges in the borate form were used to separate estrogens into two fractions: one fraction containing estrogens lacking vicinal cis-hydroxyls (Fr 1) and another containing estrogens possessing vicinal cis-hydroxyls (catecholestrogens; Fr 2). Finally, following O-trimethylsilyl ether derivatization, the estrogens were analyzed by GC/MS in the selected ion monitoring mode. Estrogens were quantitated using deuterated internal standards, which were added to the samples at the initiation of the work-up procedures. After addition to estrogen-low male human samples the standards showed good chromatographic linear response and reproducibility. A reduction in the number of steps and improvements in the robustness of the work-up procedures were achieved. The modified method described is less complex, amenable to use with commercially available SPE columns and fulfils all the reliability criteria, resulting in highly specific and accurate results.     

Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis-mass spectrometry

In this study, we use capillary electrophoresis-mass spectrometry (CE-MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE-MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).     

Isolation of water-soluble organic matter from atmospheric aerosol

The development of a solid phase extraction (SPE) method is presented, which is capable of isolating approx. 60% of the water-soluble organic compounds from aerosol samples. The aqueous extracts of the filter samples were acidified then passed through an SPE column. Four silica-based and two polymeric reversed phase columns were tested and similar recoveries of the organic carbon were found. The isolated organic matter was nearly free from inorganic ions, which are major constituents of atmospheric aerosol. This fraction accounted for a major part of the UV absorption above 250 nm and fluorescence activity of the aerosol extract. The precision of the method was tested by performing three parallel sample preparations with Oasis HLB columns. It was found that the relative standard deviation of the carbon content of the isolated organic matter was better than 7%, which indicated the reliability of the method. In the atmospheric aerosol research the newly developed sample preparation method facilitates the physical and chemical characterisation of water-soluble organic compounds without the interference of inorganic constituents.     

Optimizing the extraction and analysis of DHEA sulfate, corticosteroids and androgens in urine: application to a study of the influence of corticosteroid intake on urinary steroid profiles

A method of detecting and quantifying dehydroepiandrosterone (DHEA) sulfate, corticosteroids, and androgens has been developed. All of the compounds were first extracted from urine using solid phase extraction (SPE), enzymatically hydrolyzed, and separated into three samples using a second SPE. A DHEA sulfate sample was acetylated and re-extracted using SPE for purification before analysis. Corticosteroid samples were oxidized and re-extracted using liquid/liquid extraction for analysis. Androgen samples were acetylated and re-extracted using SPE prior to analysis. The extraction and analysis methods were investigated and optimized. Analyses were performed with gas chromatography/mass spectrometry (GC/MS) and gas chromatography/flame ionization detection (GC/FID). The entire procedure was then applied to the study of urine profiles of healthy volunteers and patients treated with corticosteroids. The results showed that the quantities of androgens found in patient urines were lower than in those of healthy volunteers. In addition, other metabolites were detected in patient urines.     

Capillary electrophoresis-mass spectrometry of proteins at medium pH using bilayer-coated capillaries

The feasibility of using noncovalently bilayer-coated capillaries for capillary electrophoresis-mass spectrometry (CE-MS) of acidic proteins was investigated using background electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000-300,000 plates) when the ionic strength was sufficiently high. Migration-time reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments showed satisfactory MS responses for studied proteins dissolved in ammonium formate (pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously suppress the MS protein signals. Evaluation of the CE-MS system was performed by analyzing a mixture of intact proteins yielding efficient separations and good-quality mass spectra. CE-MS analysis of a reconstituted formulation of the biopharmaceutical recombinant human growth hormone (rhGH) which was stored for a prolonged time, revealed one degradation product which was provisionally identified as desamido rhGH. Based on the MS responses the amount of degradation was estimated to be ca. 25%.     

Analysis of gases containing inorganic and organic compounds using a combination of a thick-film capillary column and a packed adsorption column

A gas chromatographic system consisting of one multiport valve, two (hot-wire and flame-ionization) detectors and two analytical columns (one thick-film capillary and one packed adsorption column) is used for the analysis of gas samples containing a number of inorganic compounds (hydrogen, argon, oxygen, nitrogen, carbon monoxide, and carbon dioxide) and organic compounds. Examples include samples containing hydrocarbons up to n-nonane and benzene and toluene. The system also permits the analysis of more complicated samples containing, for example, alcohols, in addition to hydrocarbons.     

Screening and analytical confirmation of sulfonamide residues in milk by capillary electrophoresis-mass spectrometry

A new methodology is proposed to automate the monitoring of sulfonamide residues in milk samples. It combines a screening unit for the total amount of sulfonamide with capillary electrophoresis-mass spectrometry (CE-MS) equipment for processing the samples containing a detectable level of sulfonamide. The screening unit consists of continuous-flow system (CFS) to precipitate the proteins connected on-line to the CE-MS equipment, in which a common characteristic ion of all sulfonamides was monitored with the MS detector by flushing the sample through the capillary. The confirmatory method is based on the purification and preconcentration of sulfonamides in a CFS unit and posterior analysis by CE-MS. The sample treatment unit was also on-line connected to the CE-MS equipment. In order to increase sensitivity, the flow rate of the sheath liquid was diminished from 0.5 to 0.2 microL.min(-1) by increasing the content in water from 0 to 50% and the formic acid from 0.5 to 1.5% in this liquid and by applying an overimposed pressure of 5 mbar during the electrophoretic separation. The method allowed the analysis of 30 samples per hour.     

The evaluation of different sorbents for the preconcentration of phenoxyacetic acid herbicides and their metabolites from soils

A procedure using alkaline extraction, solid-phase extraction (SPE) and HPLC is developed to analyze the polar herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) together with their main metabolites in soils. An ion-pairing HPLC method is used for the determination as it permits the baseline separation of these highly polar herbicides and their main metabolites. The use of a highly cross-linked polystyrene-divinylbenzene sorbent (PS-DVB) gives the best results for the analysis of these compounds. This sorbent allows the direct preconcentration of the analytes at the high pH values obtained after quantitative alkaline extraction of the herbicides from soil samples. Different parameters are evaluated for the SPE preconcentration step. The high polarity of the main analytes of interest (2,4-D and MCPA) makes it necessary to work at low flow rates (< or =0.5 mL min(-1)) in order for these compounds to be retained by the PS-DVB sorbent. A two stage desorption from the SPE sorbent is required to obtain the analytes in solvents that are appropriate for HPLC determination. A first desorption with a 50:50 methanol:water mixture elutes the most polar analytes (2,4-D, MCPA and 2CP). The second elution step with methanol permits the analysis of the other phenol derivatives. The humic and fulvic substances present in the soil are not efficiently retained by PS-DVB sorbents at alkaline pH's and so do not interfere in the analysis. This method has been successfully applied in the analysis of soil samples from a golf course treated with a commercial product containing esters of 2,4-D and MCPA as the active components.     

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018–March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.     

Novel Method for HPLC Analysis of Triterpenic Acids Using 9-Anthryldiazomethane Derivatization and Fluorescence Detection

Triterpenic acids are a group of secondary plant metabolites which are part of the cuticular waxes covering fruits, leaves, and flowers. To date, quantitative analysis of these compounds has often been conducted using high-performance liquid chromatography coupled with spectrophotometric detection or mass spectrometry; however, these methods have some major drawbacks. This paper reports a new method of analysis implementing derivatization with 9-anthryldiazomethane and fluorescence detection. The method consists of the extraction of analytes from a matrix, purification with anion exchanging SPE columns, and an optional step of the alkaline hydrolysis of triterpenic acid esters. The paper also describes a fast and easy method for the synthesis of the derivatization agent. The detection limits of the method presented are approximately 100-fold lower than in a similar method using ultraviolet spectrophotometry as the mode of detection. The recovery and repeatability of the method are at satisfactory levels.     

Glass capillary gas chromatography-mass spectrometry at high temperatures. direct analysis of free base porphyrins and metal porphyrin complexes extracted from the serpiano oil shale

This contribution describes GC/MS analysis of natural petroporphyrin extracts containing alkylporphyrins either as vanadyl complexes or as demetalated free bases. The combination of high temperature glass capillary gas chromatography with mass spectrometry allows, for the first time, direct determination of electron impact mass spectra of separated alkylporphyrins, making additional purification and derivatization unnecessary. The separation is carried out on glass capillary columns coated with the high temperature-stable medium polar, OH-terminated, polysiloxane phases PS 086 and OV-225-OH. The paper gives detailed working directions for the preparation of the high temperature GC/MS-interface, and of the high temperature stable OV-225-OH columns (max. working temperature 390°C).     

Polymer-solvent interaction parameters in polymer solutions at high polymer concentrations

Ion chromatography (IC) is widely used for the compliance monitoring of common inorganic anions in drinking water. However, there has recently been considerable interest in the development of IC methods to meet regulatory requirements for analytes other than common inorganic anions, including disinfection byproduct anions, perchlorate, and haloacetic acids. Many of these new methods require the use of large injection volumes, high capacity columns and analyte specific detection schemes, such as inductively coupled plasma mass spectrometry or postcolumn reaction with UV-Vis detection, in order to meet current regulatory objectives. Electrospray ionization mass spectrometry (ESI-MS) is a detection technique that is particularly suitable for the analysis of permanently ionized or polar, ionizable compounds. The combination of IC with MS detection is emerging as an important tool for the analysis of ionic compounds in drinking water, as it provides increased specificity and sensitivity compared to conductivity detection. This paper reports on the application of IC-ESI-MS for the confirmation and quantitation of environmentally significant contaminants, i.e. compounds with adverse health effects which are either regulated or being considered for regulation, such as bromate, perchlorate, haloacetic acids, and selenium species, in various water samples.     

Design of online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) hyphenated systems for quantitative analysis of small organic compounds in biological matrices

Three online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method examples are presented where two different types of chromatographic columns or solvent systems were coupled to meet specific analytical objectives: (i) SPE of target analytes by restricted access media from high ionic strength urine matrix was coupled with reversed phase LC-MS/MS conditions accommodating high ionization potentials of the analytes (urinary bisphenol A and other phenolic derivatives); (ii) strong cation exchange SPE of analytes of diverse polarity and pK(a) was coupled with reversed phase LC-MS/MS analysis (urinary atrazine metabolites); (iii) pre-concentration of low pg per sample analytes by weak anion exchange SPE was hyphenated with ion pair LC-MS analysis (intracellular nucleotide triphosphate analogs). With these examples we suggest a conductive generic work flow for the development of online SPE-LC-MS methods and show how advanced commercial LC devices and software allow for the design of complex yet highly versatile analytical separation systems suited to the unique physicochemical properties of the target analytes.     

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.     

Purification and quantitative analysis of urinary prostanoids in human and in rat by packed and capillary gas chromatography-negative-ion chemical-ionization mass spectrometry

We describe a method for the quantitative analysis of prostaglandin (PG) E2 and the major urinary metabolites PGI2 and thromboxane (Tx) A2 in human and in rat by combined gas chromatography and negative-ion chemical-ionization mass spectrometry. The procedure is based on the sequential use of small columns with distinct properties combined with a thin-layer chromatography step, for the extraction and the purification of urinary prostaglandins. The compounds are then analysed as their pentafluorobenzyl ester-O-methyloxime-trimethylsilyl ether derivatives, using either packed or capillary columns. Deuterated analogues are used as internal standards. The method was established by using tritiated prostaglandins covering the extremes of polarity in order to optimize the recovery of prostanoids as well as the quality of the chromatograms and spectra. The overall recovery was 24%. Standard curves were obtained by the same procedure and found to be reproducible, with a maximal day-to-day variation of +/- 5%. The relatively simple approach required for the sequential extraction and purification of prostaglandins on small columns of distinct properties, combined with the highly specific and highly sensitive method of detection, places this procedure among the most reliable method for measuring urinary prostanoids in both humans and animals. In addition, the procedure is faster than classical approaches and necessitates smaller amounts of samples and solvents.     

Evaluation of microchip capillary electrophoresis with external contactless conductivity detection for the determination of major inorganic ions and lithium in serum and urine samples

The determination of inorganic ions in clinical samples in less than 90 seconds was demonstrated for microchip capillary electrophoresis using capacitively coupled contactless conductivity detection (C(4)D). Bare electrophoresis chips were used in combination with external electrodes which were part of the chip holder. In order to achieve the required selectivity and sensitivity, an optimization of the electrode layout was carried out. Limits of detection (LOD) of 1 microM for K(+), 1.5 microM for Ca(2+), 3 microM for Na(+), 1.75 microM for Mg(2+) and 7.5 microM for Li(+) were achieved. The determination of inorganic cations (NH(4)(+), K(+), Na(+), Ca(2+), Mg(2+)) and anions (Cl(-), NO(3)(-), SO(4)(2-), phosphate) in blood serum and urine samples was possible in one common electrolyte solution containing 15 mM L-arginine, 10.75 mM maleic acid and 1.5 mM 18-crown-6 at pH 5.90 by simply switching the separation voltage from positive to negative polarity. Lithium, present at significant levels when used for therapeutic purposes, can also be determined in blood serum using a slightly modified background electrolyte solution.     

Optimization of antibiotic analysis in water by solid-phase extraction and high performance liquid chromatography–mass spectrometry/mass spectrometry

This paper describes the development of an optimized method based on solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) for the simultaneous analysis of ten antibiotic compounds including tetracyclines, sulfonamides, macrolides and quinolones. LC–MS/MS sensitivity has been optimized by alterations to both LC and MS operations. Of the two high resolution columns tested, Waters Symmetry C₁₈ endcapped and Agilent Zorbax Bonus-RP, the latter was found to show better performance in producing sharp peaks and clear separation for most of the target compounds. Optimization of the MS fragmentation collision and cone energy enhanced the peak areas of the target analytes. The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while methanol was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was shown to enhance the recovery of the compounds. To ensure a satisfactory recovery, the flow rate through SPE should be maintained at ≤10 mL min⁻¹. The method was successfully applied to the analysis of antibiotics from environmental water samples, with concentrations being <LOD in tap water, between <LOD to 28 ng L⁻¹ in river water and between <LOD to 230 ng L⁻¹ in sewage effluent.     

高效毛细管电泳在核酸、蛋白质分析中的新进展

高效毛细管电泳以其分离效率高，分析速度快，样品和试剂用量少，易于实现自动化等优点，在核酸、蛋白质等生物样品的分析方面发挥着重要的作用并具有巨大的潜力。本文介绍了近两年来高效毛细管电泳技术的进展，特别是PCR／CE、CE／MS以及电泳芯片技术等方面的新发展，并综述了高效毛细管电泳在核酸、蛋白质分析方面的应用，同时对其前景进行了展望。