LC-HRMS Method Research Articles

Targeted and untargeted screening of new psychoactive substances (NPS) and classical drugs of abuse in Paris using hair testing: A 10-years study (2012–2021)

To detect and quantify NPS and conventional drugs of abuse (DoA) between 2012 and 2021 in high-risk populations of Paris area, and to describe their characteristics using hair testing based approch and a combination of targeted and untargeted screening by LC-MSMS and LC-HRMS. In all, 939 hair samples collected in addiction ( n = 229, 24%) or intoxications ( n = 337, 36%) settings were segmented whenever possible (A: 0–2 cm; B: 2–4 cm; C: 4–6 cm) and analyzed using a previously published LC-MS/MS and LC-HRMS methods dedicated to the screening of DoA and NPS in hair. Briefly, 20 mg of hair was washed with dichloromethane, dried at room temperature and pulverized. It was hydrolyzed in phosphate buffer (pH 5.0, 95 °C, 10 min) and extracted using hexane/ethyl acetate and chloroforme/isopropanol. Separation were performed in a gradient mode using Hypersil PFP Gold column (100 mm × 2.1 mm, 1.9 μm) for targeted screening and Phenyl hexyl column (100 mm × 2.1 mm, 1.9 μm) for untargeted screening. Triple Quad TSQ Altis in MRM mode and Q Exactive Orbitrap in ddMS2 mode (both from ThermoFisher®) were used as detection systems. NPS were detected 1551 times in 288 cases. Their prevalence was 31% and was lower than that of amphetamines (34%), cocaine (42%) or licit/illicit opioids (51%) measured in the same populations. Consumers were predominantly male (68%) with a median age of 30 years. 51 different NPS were detected with 1 to 15 NPS being consumed per patient within the same period: ketamine (n = 84, 54 pg/mg median), mephedrone (26, 100), dextromethorphan (16, 100), normephedrone (9, 1630), 4-MEC (6, 205), methylone (5, 20), ethylphenidate (5, 9), 3-MMC (5, 330), flubromazepam (4, 192), etizolam (4, 5), α-PVP (4, 67), α-PHP (4, 5895), N-ethylpentylone (4, 50), 5-APB (3, 255), 3-FPM (3, 583), harmine (2, <1), mitragynine (2, <1), MDPV (2, 55), pentylone (2, 9), 2F-DCK (2, 3260), O-PCE (2, 20), 4-FA (2, 560), m-CPP (2, 569), 5F-AKB48 (n = 2, 16), 5F-PB-22 (2, 12), DMT (2, 7), 5-MeO-DMT (2, 5), 3/4-MeO-PCP (2, <1), and one case respectively for N-methyl-2-AI (326 pg/mg), 5-EAPB (60), clonazolam (24), metizolam (4), 4F-PHP (<1), 4-MePPP (10,000), butylone (68), ethcathinone (23,292), DCK (1500), MXE <1), ephenidine (190), 25C-NBOme (2), 25I-NBOme (15), AB-FUBINACA (22), ADB-PINCACA (10), AM-2201 (10), BB-22 (10), CP 47497-C8 homolog (< 1), JWH-019 (27), JWH-200 (13), JWH-250 (40). 4-HO-MiPT and isopentedrone were not quantified (lack of reference standards). Hair concentrations have allowed to distinguish occasional from chronic users. To our knowledge, this is the most comprehensive study based on hair testing demonstrating the use of different types and levels of designer drugs in Paris metropolis from 2012 to 2021. Their prevalence appears to be significant and understated as they are not systematically sought by laboratories.

Identification and quantitation of kratom in the hair of a regular user of substances purchased on the darkweb

Kratom is a natural substance with psychostimulant properties obtained from the Mitragyna speciosa species. Used today as a recreational drug, the leaves are either chewed, encapsulated, smoked or ingested as an infusion with lemon juice and sugar. Mitragynin, one of the 40 alkaloids identified in the plant, is found in 0.2% of the leaves and is considered a more potent analgesic than morphine. We present hair analysis of a 27-year-old man who self-reported daily consumption for five years of about 20 g of kratom, purchased on the darkweb, coupled with regular consumption of cannabis, GABAergic drugs and sporadically JWH-122. He donated voluntarily some hair samples. After receiving the procedure, he took himself a 3 cm long strand of brown hair, 2 months after his last consumption of JWH-122 and sent by postal way. Sample preparation was done using a published method (Fabresse et al. Drug Test Anal 2019;11:697–708). The hair strand was cut in two segments of around 1.5 cm. After decontamination, 50 mg of hair sample were incubated in phosphate buffer (pH 5.0). Liquid-liquid extraction (LLE) was performed using a mixture of heptane/ethyl acetate (v/v; 90/10) and carbonate buffer at pH 9.7. After agitation and centrifugation, a second LLE was performed by dichloromethane/isopropanol (v/v; 80/20). Organic phases were evaporated and of the dry residue was reconstituted in a mixture of water/ammonium formiate/formic acid. The screening method used an ultra-high performance liquid chromatography coupled to a benchtop Orbitrap (Q-Exactive, Thermo) operating in data-dependent acquisition mode (DDA). The same extract was then submitted to a targeted analysis by LC-MS/MS for quantitation (LCQ TSQ Vantage XP triple-quadrupole mass spectrometer, Thermo). The LC-HRMS method led to the identification of mitragynin and JWH-122. Mitragynin was quantified at 2235 pg/mg in the proximal part (A) and at 1717 pg/mg in the distal part (B). The metabolite of mitragynin, the 7-hydroxymitragynin was not identified. In parallel, using the same method, was positively quantified the JWH-122 in both hair segments (69 pg/mg in A; 103 pg/mg in B). The measured concentrations of kratom are compatible with the only concentrations described in the literature in a regular user (3 g/d) (Meier et al. Drug Test Anal 2020;12:280–4). In this case, mitragynin was measured in hair samples taken from different regions of the scalp with concentrations ranging from 1054 pg/mg to 2244 pg/mg. About the synthetic cannabinoid, the 27-year-old male self-reported a punctual JHW-122 consumption (20–40 mg/10 consecutive days). The values measured are also in accordance with the JWH-122 concentrations found in the literature based on eight hair samples from frequent users of THC-containing products or chronic abusers of classical drugs (range: 7–2800 pg/mg) (Salomone et al., Drug Test Anal 2014; 6(1–2), 126–134). Hair therefore appears to be a suitable biological matrix for the diagnosis and monitoring of kratom consumption.

Fast methods based on mass spectrometry for peptide identification. Application to sex determination of human remains in tooth enamel

Two new fast methods are proposed for the first time to assign biological sex to prehistoric human remains. Both methods are supported on sexually dimorphic amelogenin protein fragments (AMELX and AMELY) analysis in tooth enamel. The first one is based on flow injection analysis, electrospray ionization and high-resolution mass spectrometry (FIA-ESI-HRMS) with a run time of three minutes per sample. The second method is based on tandem mass spectrometry (FIA-ESI-MS/MS) with a low-resolution mass spectrometer. In this case, run time is one minute per sample.When FIA-ESI-HRMS was used, two accurate mass-to-charge ratios, corresponding to the diprotonated ions of the molecular species [M + 2H]2+of both peptides, and 6 MS/MS transitions, 3 characteristics of peptide specific to the Y isoform of amelogenin and 3 to the X, were selected for identification purposes. In the FIA-ESI-MS/MS method, 6 MS/MS transitions, 3 characteristics of peptide specific to the Y isoform of amelogenin and 3 to the X, were measured. In both cases, no separation step is carried out once the sample is injected into the system.The two methods were applied to a set of 16 tooth samples from prehistoric human remains. The results obtained in the sex determination with the rapid methods were confirmed using a liquid chromatographic based method (LC-HRMS). Results were in complete agreement among methods.A very important increase of sample throughput was obtained with the new proposed methods. When the LC-HRMS method was used, time between sample injections was 101.4 min (run time, 100 min; time require for injection, 1.4 min). When the FIA-ESI-HRMS and FIA-ESI-MS/MS methods were used, in this time (101.4 min) it was possible to analyze 23 and 48 samples, respectively. Moreover, the possibility to assign sex using low-resolution mass spectrometers means that a greater number of laboratories could perform AMEL analysis because the cost of the instrumentation is reduced.

A new on-line SPE LC-HRMS method for the analysis of Perfluoroalkyl and Polyfluoroalkyl Substances (PFAS) in PM2.5 and its application for screening atmospheric particulates from Dublin and Enniscorthy, Ireland

A sensitive analytical method has been developed and validated for the determination of 16 polyfluorinated alkyl substances (PFAS) in fine airborne particulate matter (PM2.5) using on-line solid phase extraction (SPE) coupled with liquid chromatography (LC) – negative electrospray ionisation high resolution mass spectrometry (−) ESI-HRMS. On-line SPE allows simultaneous sample clean-up from interfering matrices and lower limits of detection (LODs) by injecting a large volume of sample into the LC system without compromising chromatographic efficiency and resolution. The method provides LODs in the range 0.08–0.5 pg/mL of sample extract allowing detection of selected PFAS in aerosol particles at low fg/m3 level and showed good tolerance to the considered PM matrix. The validated method was applied for analysis of PFAS in ambient PM2.5 samples collected at two urban locations in Ireland, i.e., Enniscorthy and Dublin. Several PFAS were observed above the detection limit, including perfluorobutyrate (PFBA), perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorobutanesulfonic acid (L-PFBS) and perfluorononanoic acid (PFNA), as well as fluorotelomer sulfonates: 4:2 FTS, 6:2 FTS and 8:2 FTS. The results indicate that some toxic PFAS, such as PFOS and PFOA, are still detected in the environment despite being phased out from production and subject to restricted use in the EU and USA for more than two decades. Observation of fluorotelomer sulfonates (4:2 FTS, 6:2 FTS and 8:2 FTS, which are used as alternatives for legacy PFOA and PFOS) in ambient PM2.5 samples raises a concern about their persistence in the atmosphere and impact on human health considering emerging evidence that they could have similar health endpoints as PFOA and PFOS. To our knowledge, this is the first study to identify PFAS in ambient PM2.5 at urban locations in Ireland and also the first study to detect 4:2 and 8:2 fluorotelomer sulfonates in atmospheric aerosol particles.

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS.

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

Plasma Polyamine Biomarker Panels: Agmatine in Support of Prostate Cancer Diagnosis.

Prostate cancer is the most frequent malignant tumour among males (19%), often clinically silent and of difficult prognosis. Although several studies have highlighted the diagnostic and prognostic role of circulating biomarkers, such as PSA, their measurement does not necessarily allow the detection of the disease. Within this context, many authors suggest that the evaluation of circulating polyamines could represent a valuable tool, although several analytical problems still counteract their clinical practice. In particular, agmatine seems particularly intriguing, being a potential inhibitor of polyamines commonly derived from arginine. The aim of the present work was to evaluate the potential role of agmatine as a suitable biomarker for the identification of different classes of patients with prostate cancer (PC). For this reason, three groups of human patients—benign prostatic hyperplasia (BPH), precancerous lesion (PL), and prostate cancer (PC)—were recruited from a cohort of patients with suspected prostate cancer (n = 170), and obtained plasma was tested using the LC-HRMS method. Statistics on the receiver operating characteristics curve (ROC), and multivariate analysis were used to examine the predictive value of markers for discrimination among the three patient groups. Statistical analysis models revealed good discrimination using polyamine levels to distinguish the three classes of patients. AUC above 0.8, sensitivity ranging from 67% to 89%, specificity ranging from 74% to 89% and accuracy from 73% to 86%, considering the validation set, were achieved. Agmatine plasma levels were measured in PC (39.9 ± 12.06 ng/mL), BPH (77.62 ± 15.05 ng/mL), and PL (53.31 ± 15.27 ng/mL) patients. ROC analysis of the agmatine panel showed an AUC of 0.959 and p ≤ 0.001. These results could represent a future tool able to discriminate patients belonging to the three different clinical groups.

Analytical documentation of an Arabian horse fatality related to Oenanthe crocata poisoning

Analytical detection of Oenanthe crocata toxins in biological samples is challenging because of their instability, the lack of commercially available standards and the exceptionally low detection of these molecules using mass spectrometry. This work aims to report the used analytical methods that allowed identification of the main plant toxins in biological samples from an equid (an Arabian horse) fatality related to hemlock water dropwort (Oenanthe crocata Linnaeus) intake. Using both LC-DAD and LC-HRMS methods allowed identification (i) of oenanthotoxin in roots found on the site, root fragments found in the stomach, stomach content, kidney, and liver, and (ii) of the hydrogenated metabolite of oenanthotoxin (2,3-dihydro-oenanthotoxin) in roots found on the site, root fragments found in the stomach, stomach content, kidney, liver and spleen. Reported analytical data about Oenanthe crocata toxins can be useful for identification of the ingested plant and for supporting a poisoning diagnosis in such cases.

Differentiation of Common IGF-1 Variants Using HRMS COM Determination with Follow-Up MS/MS Verification.

Insulin-like growth factor 1 (IGF-1), a peptide hormone regulator of growth hormone (GH), has common variants with differing functionality. These variants are a result of single amino acid changes in the peptide that can lead to significant changes in the resulting protein. The standard method of evaluating any of these variants is by using tandem mass spectrometry (MS/MS) methods. A novel method has been developed to evaluate some variants solely by high-resolution mass spectrometry (HRMS) of the intact peptide by calculating the center of mass (COM) of the [M+7H]+7 isotopic distribution. This has allowed differentiation between the nonfunctional V44M variant and the A67T/A70T functional variants without the need for MS/MS. However, MS/MS is still needed to differentiate between the A67T and A70T variants. In this chapter we outline the LC-HRMS method for IGF-1 analysis with the inclusion of COM calculations and subsequent MS/MS differentiation.

Bioactive compounds in the algae of Kappaphycus alvarezii from Belang waters, Southeast Minahasa Regency

Algae Kappaphycus alvarezii is one of the leading commodities from fisheries and marine sectors in Indonesia. This algae species has many benefits in the fields of marine biology, chemistry, pharmacy, and pharmacology, as well as an excellent nutritional content for health. It is cultivated in the coastal waters of Minahasan Peninsula, especially in Buku Village, Belang District, Southeast Minahasa Regency. This study was carried out to analyze bioactive compound in the algae. Samples of 2-weeks old were taken directly from the cultivation area. Screening test was done qualitatively while an advanced test was done using LC-HRMS method. The results showed 4 bioactive compounds detected in the sample, they are alkaloids, tannins/phenolics, saponins, and flavonoids. From the advanced test, it was found 10 major compounds in the sample, i.e. (2-ethylhexyl) phthalate, phthalic acid, D-(+)-proline, maraniol, stigmatellin Y, Pentyl isopentyl phthalate, methyl thiofentanyl, phenylethanolamine, acetyl hexamethyl tetralin, and fucoxanthin.Indonesian title: Senyawa bioaktif pada alga Kappaphycus alvarezii dari perairan Belang, Kabupaten Minahasa Tenggara

Chemical composition and biological activities of propolis samples from different geographical regions of Turkey

• The chemical and volatile content of 39 Turkish propolis were determined. • They exhibited significant cytotoxicity against MDA-MB-231, HeLa, A549, PC-3. • NO production was suppressed on LPS-activated RAW 264.7 cells. • Most of the propolis extracts exhibited more than 80 % DPPH scavenging activities. • Propolis indicate antiviral potential against IBV and no toxic effects in ovo model. Propolis is a natural product with many biological activities, and it has been used in many industrial areas, such as pharmaceuticals, food and cosmetics. Thus, in this study, we aim to evaluate the chemical content, antioxidant, antiviral activities, cytotoxicity and the iNOS of propolis samples collected from 39 different regions in Turkey. Phenolic, flavonoid and triterpene profiles were determined using the LC-HRMS method. The main phenolic compounds identified were flavonoids, including diosmetin, rhamnocitrin, isosakuranetin, naringenin, chrysin, 3- O -methyl-quercetin and acacetin, the main phenolic acid was found as caffeic acid. The major triterpene compounds were also detected as oleanolic acid and hederagenin. The composition of volatile compounds in the propolis samples was examined using GC–MS. α -Pinene, β -pinene and limonene were found as the main compounds in most of the samples. Cytotoxic activities against MDA-MB-231, PC-3, A549, HeLa and a non-tumor cell line HEK293 were performed using the MTT method. The iNOS was also determined using LPS induced RAW 264.7 macrophage cells. Propolis samples exhibited significant cytotoxic and nitric oxide inhibition activities with a significant range of IC50 values. The total antioxidant/phenolic capacities in TR equivalents of propolis samples were assayed by DPPH, CUPRAC, FRAP, and Folin methods. The total phenolics content in the studied propolis extracts were found as between 0.96 and 13.53 mmol TR/g. TAC of propolis samples range with respect to the CUPRAC and FRAP methods were found from 0.71 to 8.24 and 0.21 to 2.27 mmol TR/g, respectively. Most of the propolis extracts exhibited more than 80 % DPPH scavenging activities, almost similar or higher than the positive controls. Antiviral activities of the samples were measured as virucidal activity by in ovo . All propolis samples promise for the significant inhibition of avian coronavirus. To our knowledge, this study is the first detailed study in the literature that includes the chemical content and biological activities of Turkish propolis collected from different geographical regions.

Development and validation of an LC-HRMS method for the determination of pyrrolizidine alkaloids and quinolones in honey employing a simple alkaline sample dilution

Development and validation of an LC-HRMS method for the determination of pyrrolizidine alkaloids and quinolones in honey employing a simple alkaline sample dilution

The Development of a Liquid Chromatography High-Resolution Mass Spectrometric Method for Apixaban Quantification in Dried Plasma Spots in Parallel Reaction Monitoring Mode

This work aimed at developing and validating a rapid, sensitive, and robust method of liquid chromatography with high-resolution mass spectrometry (LC–HRMS) in parallel reaction monitoring (PRM) mode for apixaban quantification in dried plasma spots (DPSs) with a simple extraction procedure. A 25 µL sample of human plasma was placed onto Whatman 903 Protein Saver Cards and allowed to dry; 3.2 mm diameter disks were cut out from DPSs using a puncher, and 100 µL of a working internal standard solution was added to each sample. After this, they were vortexed on a shaker for 15 min at 800 rpm and 40 °C and quick centrifugation (10,000× g, 10 s), and then the extracts were transferred into a 300 µL vial for LC–HRMS. Data were acquired in PRM mode via detection of all target product ions with 10 ppm tolerance. Total analysis time was 5 min. The LC–HRMS method was validated for the 10–400 ng/mL range with R2 &gt; 0.99. Within this range, intra- and interday variability of precision and accuracy was &lt;10%, and recovery was 69.7–85.1%. Apixaban was stable after brief storage at room temperature, and at 4 °C for up to a month. The method development and validation results proved that this LC–HRMS assay of apixaban in DPSs is selective and robust.

Understanding sweetness of dry wines: First evidence of astilbin isomers in red wines and quantitation in a one-century range of vintages

Astilbin (2R, 3R) was recently reported to contribute to wine sweetness. As its aglycon contains two stereogenic centers, three other stereoisomers may be present: neoisoastilbin (2S, 3R), isoastilbin (2R, 3S), and neoastilbin (2S, 3S). This work aimed at assaying their presence for the first time in wines as well as their taste properties. The isomers were synthesized from astilbin and purified by semi-preparative HPLC. With the four stereoisomers, a sweet taste was perceived whose intensity varied with the configuration. Their content was assayed by developing a UHPLC-Q-Exactive method. The method was applied to screen astilbin and isomers in various wines, especially in different vintages from the same estate. While young wines contained higher concentrations of astilbin than the old ones, the concentrations of the other isomers, mainly neoastilbin, were higher in the old wines, suggesting their formation over time.

Liquid Chromatography–High-Resolution Mass Spectrometry-Based Target and Nontarget Screening Methods to Characterize Film-Forming Amine-Treated Steam-Water Systems

Film-forming amine products (FFAP) are frequently used as corrosion inhibitors in steam-water systems. To have a better understanding of their behavior in the treated system, a liquid chromatography coupled to the high-resolution mass spectrometry (LC–HRMS) method was developed. This method enabled the concurrent targeted quantitative analysis of the film-forming amine oleyl propylenediamine (OLDA) and the nontarget screening (NTS)-based analysis of potential degradation products of FFAP or other organic matter in the system. The method was applied to samples collected from an FFAP-treated industrial steam-water system. OLDA was detected at a concentration of 0.027 mg/L after the FFAP dosing in the low-pressure steam drum. A feature (a combination of mass-to-charge ratio and retention time associated with a signal intensity) prioritization workflow was designed and applied to the NTS results to focus on the potential thermal degradation products of FFAP that could affect the studied steam-water system. Six features were prioritized from a total of 4596 detected features. Structural elucidation of these features was attempted based on mass spectrometry fragmentation data. One of the prioritized features was successfully identified as 2,2′-(tridecylimino)diethanol, presumably a degradation product of the FFAP emulsifier. Because of the chemical structure of the molecule, 2,2′-(tridecylimino)diethanol was considered as noncorrosive to the system. Overall, the developed LC–HRMS method facilitated the efficient analysis of OLDA with a limit of quantification of 0.002 mg/L and a comprehensive monitoring of organic matter across the steam-water system, including potential degradation products of the dosed FFAP.

Metabolism Study of N-Methyl 2-Aminoindane (NM2AI) and Determination of Metabolites in Biological Samples by LC-HRMS.

Since the widespread diffusion of new psychoactive substances, forensic laboratories are often required to identify new drugs and their metabolites for which information or reference standards are lacking. We performed a study on N-methyl-2-aminoindane (NM2AI) metabolism in silico and in vivo, in order to identify the main metabolites to be screened in the different biological samples. We performed the in silico metabolism prediction of NM2AI using MetaSiteTM software and subsequently verified the presence of metabolites in the blood, urine and hair of mice after NM2AI administration. The samples were analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with a benchtop Orbitrap Exactive mass detector. This allowed the evaluation of the agreement between software prediction and experimental results in biological samples. LC-HRMS analysis identified seven main metabolites in the urine. They were identified, by their accurate masses and fragmentation patterns, as 2-aminoindane (2AI), two hydroxy-2AI and four hydroxy-NM2AI; one of the hydroxy-NM2AI and one of the hydroxy-2AI underwent also to conjugation. NM2AI and 2AI were also detected by LC-HRMS in the hair and blood. Based on these findings, we developed an LC-HRMS method for the screening of NM2AI and metabolites in urine, blood and hair samples. This can be of primary effectiveness to uncover the abuse of NM2AI and related possible intoxications.

Riboflavin degradation products; combined photochemical and mass spectrometry approach

Flavins are compounds of important biological functions due to their photochemical and photophysical properties. We report photochemical properties in acetonitrile and mass spectrometric data of riboflavin analogue, 3-benzylriboflavin. In photochemical experiment there are formed two main (3-benzyllumichrome and 3-benzyllumiflavin) and five additional photoproducts, most of them of isoalloxazine structure. Photoproducts, identified by mass spectra, have been analyzed in comparison to the respective MS fragmentation patterns. Results for 3-benzylriboflavin was compared to those obtained for riboflavin and 2-thioriboflavin. EI-MS, ESI-MS and LC-HRMS methods have been used, the latter combined with the ESI techniques. The MS details combined with the photolysis data display some similarities, indicating the relative stability of some products formed upon UV-VIS irradiation and zfragmentation.

A high-throughput assay for the quantification of intact Insulin-like Growth Factor I in human serum using online SPE-LC-HRMS

Quantification of IGF-I is relevant in both doping control as a biomarker of growth hormone (GH) misuse in sports, and in the clinical field for longitudinal follow-up of patients with disorders related to the GH axis. Currently, better standardization of IGF-I measurements using mass spectrometry is in our best interest as it would enable long-term monitoring of an athletes’ IGF-I levels by its addition to the Athlete Biological Passport (ABP).Here, a simplified and rapid top-down LC-HRMS method for quantification of IGF-I in human serum is presented. A ten-minute precipitation-based offline sample preparation is combined with online sample clean-up and separation on a conventional LC, resulting in a total runtime of nine minutes in between injections. The method was validated in the relevant range of 50–1000 ng/mL for the following parameters: linearity, precision, bias, Limit Of Quantification (LOQ), carry-over, selectivity, recovery and ion suppression. As proof of concept, the presented LC-HRMS assay was compared with results from a previous inter-laboratory study on intact IGF-I quantification using four human GH administration samples. It was additionally compared with the IDS-iSYS immunoassay using 47 athlete serum samples, showing good overall agreement with a slight positive bias of 24.2 ng/mL for the LC-HRMS assay at a mean sample concentration of 234 ng/mL. Also, a discrepancy between commercially available IGF-I reference material for the calibration of quantitative assays is discussed. This is of importance if LC-MS assays for IGF-I are to be harmonized.

Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry.

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5–80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0–24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0–24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

A novel LC-HRMS method reveals cysteinyl and glutathionyl polysulfides in wine

A novel ultra high pressure liquid chromatography combined with high resolution mass spectrometry (UHPLC-HRMS) method was developed to study glutathionyl and cysteinyl polysulfides in wine. Different HPLC columns were investigated in order to optimise the chromatographic resolution of the polysulfide standard mixtures synthesised in-house. The optimisation of the chromatographic conditions when trying to separate glutathionylated and cysteinylated species containing from 3 to 5 sulfur atoms proved particularly challenging, with the cationic exchange column IonPac CS12A-MS resulting to be the best column for this task.The synergistic application of the newly developed methods together with the synthesised reference standard mixtures allowed the identification and the detection of 11 different glutathionyl and cysteinyl polysulfides. Moreover, analysing 15 young white wines was possible to confirm the presence of GSSSG in wine (GS = glutathione). More importantly, this study allowed for the first identification of several symmetric and asymmetric new polysulfides, namely: GSSSSG, CSSSC (CS = cysteine), CSSSSC, CSSSG, and CSSSSG. These molecules have not previously been identified in wine, raising the question on their biogenesis and role on wine quality.

Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.