Application of CE–ICP–MS and CE–ESI–MS in metalloproteomics: challenges,developments, and limitations

Application of capillary electrophoresis (CE) as a high-resolution separation technique in metalloproteomics research is critically reviewed. The focus is on the requirements and challenges involved in coupling CE to sensitive element and molecule-specific detection techniques such as inductively coupled plasma mass spectrometry (ICP–MS) or electrospray ionisation mass spectrometry (ESI–MS). The complementary application of both detection techniques to the structural and functional characterisation of metal-binding proteins and their structural metal-binding moieties is emphasised. Beneficial aspects and limitations of mass spectrometry hyphenated to CE are discussed, on the basis of the literature published in this field over the last decade. Recent metalloproteomics applications of CE are reviewed to demonstrate its potential and limitations in modern biochemical speciation analysis and to indicate future directions of this technique.     

LC–MS–MS Determination of Rotenone, Deguelin, and Rotenolone in Human Serum

For the first time we report a rapid and sensitive LC–MS–MS method for quantification of rotenone, deguelin, and rotenolone in human serum. The analytical procedure involves extraction with ethyl acetate without further clean-up. The active ingredients were separated on a C₈ reversed-phase column by isocratic elution. Eleven simultaneous transitions of precursor ions were monitored. Excellent selectivity and sensitivity enables quantification and identification of low levels of rotenoids (LOD 2 ng mL^?1, LOQ 5 ng mL^?1) in human serum.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

Simultaneous measurement of endogenous cortisol,cortisone, dehydroepiandrosterone,and dehydroepiandrosterone sulfate in nails by use of UPLC–MS–MS

Steroid hormone concentrations are mostly determined by using different body fluids as matrices and applying immunoassay techniques. However, usability of these approaches may be restricted for several reasons, including ethical barriers to invasive sampling. Therefore, we developed an ultra-performance LC–MS–MS method for high-throughput determination of concentrations of cortisol, cortisone, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEAS) in small quantities of human nails. The method was validated for linearity, limits of detection and quantification, recovery, intra and interassay precision, accuracy, and matrix effect. Samples from 10 adult women were analyzed to provide proof-of-principle for the method’s applicability. Calibration curves were linear (r ² > 0.999) in the ranges 10–5000 pg mg⁻¹ for cortisol, cortisone, and DHEAS, and 50–5000 pg mg⁻¹ for DHEA. Limits of quantification were 10 pg mg⁻¹ for cortisol, cortisone, and DHEAS, and 50 pg mg⁻¹ for DHEA. The sensitivity and specificity of the method were good, and there was no interference with the analytes. Mean recovery of cortisol, cortisone, DHEA, and DHEAS was 90.5%, 94.1%, 84.9%, and 95.9%, respectively, with good precision (coefficient of variation <14% for all analytes) and accuracy (relative error (%) −8.3% to 12.2% for all analytes). The median (pg mg⁻¹, range) hormone concentrations were 69.5 (36–158), 65 (32–133), 212 (50–1077), and 246 (115–547) for cortisol, cortisone, DHEA, and DHEAS, respectively. This method enables measurement of cortisol, cortisone, DHEA, and DHEAS in small quantities of human nails, leading to the development of applications in endocrinology and beyond.     

Simultaneous Determination of Fluoroquinolones, Tetracyclines and Sulfonamides in Chicken Muscle by UPLC–MS–MS

A rapid and sensitive analytical method for the simultaneous determination of four fluoroquinolones, four tetracyclines and six sulfonamides in chicken muscle using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS–MS) has been developed and validated. Samples were extracted with McIlvaine buffer-acetonitrile, defatted with n-hexane, and analyzed by UPLC–MS–MS. Solvent delay technique was applied in the analysis to remove the non-volatile phosphate and carry out farther on-line SPE clean-up. Satisfactory recoveries (55–110%) of all the veterinary drugs were demonstrated in 1, 10 and 20 μg kg^?1 spiked levels with the overall RSD for intra- and inter-day of 14 analytes less than 18%. The LOD and LOQ were 0.3 and 1.0 μg kg^?1, respectively. Quantitative results of 103 real samples indicated that the present method was suitable for the quantitative analysis of real samples.     

Investigation of Euphrasia rostkoviana Hayne Using GC–MS and LC–MS

Gas and liquid chromatography coupled with mass spectrometry was applied to solve difficulties and reinvestigate the serious matrix problems affecting analysis of the active compounds in Euphrasia rostkoviana Hayne. The main groups of compounds were obtained by extracting the herb stepwise with n-hexane, chloroform, ethyl acetate and methanol. Polyamide column chromatography facilitated further separation. Phenolic/flavonoid- and terpenoid-type molecules were studied by GC–MS, HPLC and LC–MS–MS. The β-sitosterol content of the herb was determined by gas chromatography with flame ionisation detection (GC-FID). Caffeic acid, chlorogenic acid, coumaric acid and flavonoid glycosides of apigenin, luteolin, rhamnetine (hexoside), kaempferol (both hexoside and rutinoside) and quercetin (rutinoside) were identified in the fractions of the methanolic extract.     

LC–MS–MS Determination of Stabilizers and Explosives Residues in Hand-Swabs

The contribution of explosive trace detection in samples from the hands of suspects has been fundamental in several forensic cases involving terrorists. This paper describes a method for the rapid extraction and unequivocal confirmation of some high potential explosives (trinitrotoluene, cyclotrimethylenetrinitramine, pentaerythritol tetranitrate, nitroglycerin) and two stabilizer (diphenylamine and ethylcentralite) residues in hand-swabs using liquid chromatography–tandem mass spectrometry. The extraction procedure of the analytes from the swabs is realized by solvent elution and the extracts are directly analyzed. Recoveries from spiked swabs range from 78 to 96%; the limits of quantification are between 0.04 and 1.8 ng injected and the inter-day method precision is less than 15%. The developed procedure was applied to the detection of explosives traces in samples after handling tests.     

Quantitative determination of cationic modified polysaccharides on hair using LC–MS and LC–MS–MS

Cationic polysaccharides containing N-hydroxypropyl-N,N,N-trimethylammonium substituents are widely used as conditioning agents for hair-care products. A sensitive method has been developed for the quantitation of these polymers. After acidic extraction from hair the polysaccharides are hydrolyzed using trifluoroacetic acid. The cationic monoglycosides are determined using liquid chromatography–tandem mass spectrometry (LC–MS–MS). The developed method is independent of hair treatment. Even hair cut from test persons after customary hair wash can be analyzed. After treatment of natural and bleached hair tresses using a real-life treatment procedure 180 g and 300 g of polymer per gram hair were quantified, respectively. Additionally the fragmentation mechanism of the cationic N-hydroxypropyl-N,N,N-trimethylammonium group during electrospray ionization was investigated. A mass loss of 60 Da in combination with loss of a single charge is observed and associated with cleavage of trimethylamine and a proton. It is assumed that this process is promoted by the anionic counter-ion which might be hydroxide in an aqueous environment.     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Simultaneous Determination of Levodopa, Benserazide and 3-O-Methyldopa in Human Serum by LC–MS–MS

For the first time a sensitive, specific and rapid LC–MS–MS assay is presented for the simultaneous determination of levodopa (L-DP), 3-O-methyldopa (3-OMD) and benserazide (BSZ) in human serum. The three compounds were extracted from human serum by protein precipitation followed by dilution of the supernatant with aqueous formic acid. In serum, linearity was observed between 50 and 1,000 ng mL^?1 of L-DP, 3-OMD and BSZ, respectively. Intra-day and inter-day RSD values were below 10.56 and 6.22% at concentrations of 120, 360 and 720 ng mL^?1. The presented method showed excellent specificity and sensitivity compared with other methods reported. It was applied to a pharmacokinetic study and demonstrated its applicability to pre-clinical and clinical pharmacological research.     

Assessment of robustness for an LC–MS–MS multi-method by response-surface methodology,and its sensitivity

Sensitive, fast, and robust multi-methods are required for the surveillance of the contamination of the drinking water resources by organic trace contaminants. In the present work an alternative strategy using response surface methodology (RSM) was applied for assessment of the robustness of a LC–MS–MS multi-method. The analytical method was optimised by means of a central composite design including six design variables. The main object was to evaluate the significance of the RSM results with regard to robustness and to the sensitivity to the mass transitions used in the multi-method. The robustness of the multi-method was represented by the curvature of the calculated response surfaces for the response value R. Furthermore, it could be demonstrated that the RSM was sensitive to changes made to the investigated data set and was able to clearly indicate the fraction of substances, which met the defined criterion for signal-to-noise-ratio.     

Validation and use of three complementary analytical methods (LC–FLS, LC–MS/MS and ICP–MS) to evaluate the pharmacokinetics, biodistribution and stability of motexafin gadolinium in plasma and tissues

Liquid chromatography–fluorescence (LC–FLS), liquid chromatography–tandem mass spectrometry (LC–MS/MS) and inductively coupled plasma–mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC–FLS method exhibited the greatest sensitivity (0.0057 μg mL⁻¹), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC–MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP–MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC–FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg⁻¹ MGd was determined by LC–FLS as follows: plasma (0.025±0.002 μg mL⁻¹), liver (2.89±0.45 μg g⁻¹), and kidney (6.09±1.05 μg g⁻¹). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC–MS/MS and ICP–MS methods. For a representative patient, ≥90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.     

Determination of 5-Hydroxytryptamine, Norepinephrine, Dopamine and Their Metabolites in Rat Brain Tissue by LC–ESI–MS–MS

A liquid chromatography–electrospray ionization tandem mass spectrometry method has been developed to perform the determination of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites, i.e., 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat brain tissue. Analytes were separated on a Thermo C₁₈ column (4.6 mm × 250 mm, 5 μm, SN: 1245575T, Thermo electron corporation, USA) with a mobile phase of 0.05% formic acid/acetonitrile (92:8 for ESI⁺, 82:18 for ESI^?, v/v) at the flow-rate of 0.8 mL min^?1. The LC system was coupled to a Waters Micromass Quattro Premier XE tandem quadruple mass spectrometer. MS acquisition of 5-HT, NE and DA was performed in positive electrospray ionization multiple reaction monitoring (MRM) mode, while negative electrospray ionization MRM mode was used to monitor their metabolites. The calibration curves were linear within the concentration range of 4–4,450 ng mL^?1 for 5-HT, 4–4,110 ng mL^?1 for NE and 4–4,100 ng mL^?1 for DA (r ≥ 0.999). The limit of quantitation was 4 ng mL^?1. 5-HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12–1,000 ng mL^?1(r ≥ 0.998) and the limit of quantitation was 12 ng mL^?1. The intra- and inter-day RSD were lower than 8.45%. The method is sensitive, fast, accurate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.     

UPLC–MS–MS Analysis of Quetiapine and Its Two Active Metabolites, 7-Hydroxyquetiapine and 7-Hydroxy-N-dealkylquetiapine, in Rat Plasma and Cerebrospinal Fluid

Quetiapine (QP) is an antipsychotic agent widely used to treat a variety of human psychotic disorders. 7-Hydroxyquetiapine (QPOH) and 7-hydroxy-N-dealkylquetiapine (QPND) are its two active metabolites. A rapid and sensitive ultra-performance liquid chromatographic–tandem mass spectrometric method has been developed for analysis of the three agents in plasma and cerebrospinal fluid (CSF) from rats. The assay was based on liquid–liquid extraction of 100-μL samples. The methods were validated for QP, QPOH, and QPND in both types of sample. Limits of detection (LOD) in plasma were 0.02, 0.01, and 0.02 ng mL^?1 for QP, QPOH, and QPND, respectively; in CSF samples the respective values were 0.02, 0.01, and 0.01 ng mL^?1. The utility of the method was demonstrated by analysis of the pharmacokinetics and CSF distribution properties of QP and its two active metabolites in the plasma and CSF in rats.     

Simultaneous determination of deoxynivalenol,zearalenone, and their major masked metabolites in cereal-based food by LC–MS–MS

Cereals and cereal-based food have often been found to be contaminated with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON), after infection of the grain with the pathogenic fungus Fusarium. Both the pathogen and the infected plants can chemically modify DON and ZON, including acetylation, glucosidation, and sulfation. Analytical strategies for detection and quantification of DON and ZON are well known and established but often fail to recognize the respective metabolites, which are, therefore, also referred to as “masked” mycotoxins. However, several masked forms are also known to be harmful to mammals. Failure to detect these could lead to significant underestimation of the toxic potential of a particular sample. To monitor the levels of DON and ZON metabolites in cereals and cereal-based food, we have developed a LC–MS–MS method capable of simultaneous determination of DON, ZON, and eight of their masked metabolites, namely deoxynivalenol-3-glucoside (D3G), 3-acetyl-deoxynivalenol (3ADON), zearalenone-4-glucoside (Z4G), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalenol-4-glucoside (α-ZG), β-zearalenol-4-glucoside (β-ZG), and zearalenone-4-sulfate (Z4S). The suitability of several cleanup strategies including C₁₈-SPE, primary and secondary amines (PSA), MycoSep push-through columns, and immunoaffinity columns was evaluated. The final method used no sample cleanup and was successfully validated for four cereal-based food matrices, namely cornflour, porridge, beer, and pasta, showing good recoveries and precision for all analytes.     

LC–MS–MS Analysis of Strictosamide in Rat Plasma, and Application of the Method to a Pharmacokinetic Study

A rapid and simple high-performance liquid chromatographic tandem mass spectrometric method has been developed and validated for analysis of strictosamide in rat plasma. Chromatographic separation was achieved on a C₁₈ column by gradient elution with mixtures of methanol, water, and acetonitrile containing 0.05% acetic acid. Digoxin was used as internal standard. Selected reaction monitoring (SRM) was used for MS quantitation. Linearity was good in the range 0.05–20 ng mL^?1 in rat plasma. The lower limit of quantitation was 0.04 ng mL^?1. The method is precise and reliable and can be applied to pharmacokinetic studies.     

Analysis and validation of perfluorinated compounds in water,sediment and fish with LC-ESI–MS/MS

The analysis of perfluorinated compounds (PFCs) in environmental matrices is challenging, as the concentrations are generally low, but the risk of contamination is high. Sample preparation is a critical step and it is necessary to minimise the possibility of contamination. In this study, we successfully applied and validated a modified ion pair extraction method to quantify PFCs in sediment and fish samples. A large volume injection method was validated and used to quantify PFCs in different water matrices. Isotope internal standard of every analyte was applied to correct matrix effects. The recoveries of the analytes were 92–106% for water matrix, 93–119% for fish matrix and 86–103% for soil matrix whereas the achieved limit of quantitation values were 1.3–14.9 ng/L for water, 0.19–0.28 μg/kg for fish and 0.14–0.41 for soil samples. Thirty-one surface water, 8 stormwater and 41 sediment samples collected all over Estonia were analysed and 4 (out of 8 analysed) PFCs were found in quantitative amount. The most frequently detected analyte perfluorobutanoic acid (PFBA) was found in 26% of the water samples with a maximum concentration of 137 ng/L.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.