Simultaneous Determination of 1-Methyltryptophan and Indoleamine 2,3-Dioxygenase Biomakers of Tryptophan and Kynurenine in Mice Tumors by HPLC–MS/MS

Indoleamine 2,3-dioxygenase (IDO), an immune checkpoint protein, can cause the depletion of tryptophan (Trp) and accumulation of its metabolite of kynurenine (Kyn) in cancer cells, and generates the immunosuppressive microenvironment that supports tumor cell growth. A novel immunoregulatory prodrug micelle based on polyethylene glycol-derivatized an IDO-selective inhibitor of 1-methyltryptophan (1-MT), PEG-Fmoc-1-MT, was developed for inhibiting the IDO activity of the conversion of Trp to Kyn in tumor microenvironments. To investigate the 1-MT distribution and Trp/Kyn ratios in mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment, a HPLC–MS/MS method for simultaneous determination of 1-MT and IDO biomakers of Trp and Kyn in mouse tumors was developed and validated. Triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple reaction monitoring at m/z 219.0?→?160.1, 205.0?→?118.2, 209.0?→?146.1 and 249.3?→?148.3 was used for determination of 1-MT, Trp, Kyn and matrine (internal standard). The method demonstrated good linearity at the concentrations ranging from 10 to 10,000 ng/mL and lower limits of quantitation of 1 ng/mL for 1-MT, Trp and Kyn, respectively. The validated method was successfully applied to 1-MT tumor biodistribution and Trp/Kyn ratio studies in 4T1 tumor bearing mice i.v. with PEG-Fmoc-1-MT prodrug micelles. The mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment exhibited higher 1-MT accumulation and lower Trp/Kyn ratio, in comparison with those of mice with 1-MT solution treatment. The developed PEG-Fmoc-1-MT prodrug micelles could be a promising IDO immunoregulatory prodrug micelles for cancer immunotherapy.

     

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.     

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.     

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.     

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.     

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

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.     

Development and validation of a LC–MS/MS method for the simultaneous determination of aflatoxins, dyes and pesticides in spices

Based on several alerts from European countries over the last years concerning spices, we have been encouraged to establish an accurate method for the determination of dyes, aflatoxins and pesticides in various types of spices using reversed-phase (RP) liquid chromatography–tandem mass spectrometry interfaced with electrospray (LC–ESI–MS/MS). A simple sample treatment procedure entailing the use of an extraction step with acetonitrile without further cleanup has been developed. A C18 column with an aqueous ammonium formate/methanol mixture as the mobile phase was used, and gradient elution was performed. Mass spectral acquisition was done in positive ion mode by applying multiple reaction monitoring of at least two fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of linearity, sensitivity, repeatability, recovery and selectivity on six kinds of spices. Satisfactory results in the majority of the cases were obtained for all analytes and matrices, with practical limits of quantitation acceptable for routine monitoring purposes. Extraction recoveries for most of the compounds ranged from 60% to 140% at spiking levels of 0.05 and 0.5 mg kg⁻¹. The applicability of the method for the simultaneous determination of dyes, aflatoxins and pesticides in several types of spices was demonstrated, and the method successfully applied to a limited number of products from the local market.     

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.     

Determination of methotrexate, 7-hydroxymethotrexate,and 2,4-diamino-N10-methylpteroic acid by LC–MS/MS in plasma and cerebrospinal fluid and application in a pharmacokinetic analysis of high-dose methotrexate

A rapid and robust method for measuring methotrexate (MTX) and its two primary metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA), was developed for use in pharmacokinetic studies of plasma and cerebrospinal fluid samples collected from infants with malignant brain tumors. Sample aliquots (100?µL) were prepared for bioanalysis of MTX and metabolites using a Waters Oasis HLB microelution solid-phase extraction (SPE) plate. Chromatography was performed using a Phenomenex Synergi Polar-RP 4 µ 75?×?2.0?mm ID column heated to 40°C. A rapid gradient elution on a Shimadzu HPLC system was used, with mobile phase A consisting of water/formic acid (100/0.1?v/v) and mobile phase B consisting of acetonitrile/formic acid (100/0.1?v/v). Column eluent was analyzed using AB Sciex QTRAP 5500 instrumentation in electrospray ionization mode. The ion transitions (m/z) monitored were 455.2?→?308.1, 471.1?→?324.1, and 326.2?→?175.1 for MTX, 7-OHMTX, and DAMPA, respectively. The method was linear over 0.0022–5.5?µM for MTX, 0.0085–21?µM for 7-OHMTX, and 0.0031–7.7?µM for DAMPA. The method was applied to the analysis of serial plasma samples obtained from infants diagnosed with malignant brain tumors receiving high-dose methotrexate and results were compared to MTX concentrations from an immunoassay based on fluorescence polarization.     

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.     

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 and sensitive LC–MS/MS determination of tetrahydrocannabinol and metabolites in human plasma

Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ?⁹-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng?mL^?1 for THC and 11-nor-?⁹-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng?mL^?1 for ?⁹-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.     

Comparison of LC and UPLC Coupled to MS–MS for the Determination of Sulfonamides in Egg and Honey

Determination of ten sulfonamides (SAs) in egg and honey has been compared using column liquid chromatography (LC) and ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS–MS). A liquid–liquid extraction with acetonitrile followed by solid-phase extraction on a Strata-X cartridge was developed for sample preparation. The analytical performance of both methods was compared applying the alternative matrix-comprehensive in-house validation approach using specially designed software InterVal?. Using UPLC the separation time was shortened about 30% reducing the run time by 8 min and a better resolution was achieved compared to LC. Due to higher peak efficiency achieved with UPLC, the decision limit values obtained by both techniques were almost equal (6.61–9.43 μg kg^?1 and 7.25–11.9 μg kg^?1 for UPLC and LC, respectively), despite the fact that in UPLC twice lower sample volumes were injected. Satisfactory and comparable recoveries (80–110%) were obtained by UPLC and LC for all the SAs, except for sulfacetamide by LC and sulfabenzamide by both methods. For a majority of the spiked compounds, UPLC gave significantly better precision.     

Rapid and sensitive HILIC–MS/MS analysis of carnitine and acetylcarnitine in biological fluids

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography–tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

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.     

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.     

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.     

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.