From a single steroid to the steroidome: Trends and analytical challenges.

For several decades now, the analysis of steroids has been a key tool in the diagnosis and monitoring of numerous endocrine pathologies. Thus, the available methods used to analyze steroids in biological samples have dramatically evolved over time following the rapid pace of technology and scientific knowledge. This review aims to synthetize the advances in steroids' analysis, from classical approaches considering only a few steroids or a limited number of steroid ratios, up to the new steroid profiling strategies (steroidomics) monitoring large sets of steroids in biological matrices. In this context, the use of liquid chromatography coupled to mass spectrometry has emerged as the technique of choice for the simultaneous determination of a high number of steroids, including phase II metabolites, due to its sensitivity and robustness. However, the large dynamic range to be covered, the low natural abundance of some key steroids, the selectivity of the analytical methods, the extraction protocols, and the steroid ionization remain some of the current challenges in steroid analysis. This review provides an overview of the different analytical workflows available depending on the number of steroids under study. Special emphasis is given to sample treatment, acquisition strategy, data processing, steroid identification and quantification using LC-MS approaches. This work also outlines how the availability of steroid standards, the need for complementary analytical strategies and the improvement of calibration approaches are crucial for achieving complete steroidome quantification.

Developments in tandem ion mobility mass spectrometry.

Ion Mobility (IM) coupled to mass spectrometry (MS) is a useful tool for separating species of interest out of small quantities of heterogenous mixtures via a combination of m/z and molecular shape. While tandem MS instruments are common, instruments which employ tandem IM are less so with the first commercial IM-MS instrument capable of multiple IM selection rounds being released in 2019. Here we explore the history of tandem IM instruments, recent developments, the applications to biological systems and expected future directions.

Advancements in the gold standard: Measuring steroid sex hormones by mass spectrometry.

Sex hormones, such as testosterone and estrogens, play an essential role in regulating physiological and reproductive development throughout the lifetime of the individual. Although variation in levels of these hormones are observed throughout the distinct stages in life, significant deviations from reference ranges can result in detrimental effects to the individual. Alterations, by either an increase or decrease, in hormone levels are associated with physiological changes, decreased reproductive capabilities, and increased risk for diseases. Hormone therapies (HTs) and assisted reproductive technologies (ARTs) are commonly used to address these factors. In addition to these treatments, gender-affirming therapies, an iteration of HTs, are also a prominent treatment for transgender individuals. Considering that the effectiveness of these treatments relies on achieving therapeutic hormone levels, monitoring of hormones has served as a way of assessing therapeutic efficay. The need for reliable methods to achieve this task has led to great advancements in methods for evaluating hormone concentrations in biological matrices. Although immunoassays are the more widely used method, mass spectrometry (MS)-based methods have proven to be more sensitive, specific, and reliable. Advances in MS technology and its applications for therapeutic hormone monitoring have been significant, hence integration of these methods in the clinical setting is desired. Here, we provide a general overview of HT and ART, and the immunoassay and MS-based methods currently utilized for monitoring sex hormones. Additionally, we highlight recent advances in MS-based methods and discuss future applications and considerations for MS-based hormone assays.

After another decade: LC-MS/MS became routine in clinical diagnostics.

Tandem mass spectrometry - especially in combination with liquid chromatography (LC-MS/MS) - is applied in a multitude of important diagnostic niches of laboratory medicine. It is unquestioned in its routine use and is often unreplaceable by alternative technologies. This overview illustrates the development in the past decade (2009-2019) and intends to provide insight into the current standing and future directions of the field. The instrumentation matured significantly, the applications are well understood, and the in vitro diagnostics (IVD) industry is shaping the market by providing assay kits, certified instruments, and the first laboratory automated LC-MS/MS instruments as an analytical core. In many settings the application of LC-MS/MS is still burdensome with locally lab developed test (LDT) designs relying on highly specialized staff. The current routine applications cover a wide range of analytes in therapeutic drug monitoring, endocrinology including newborn screening, and toxicology. The tasks that remain to be mastered are, for example, the quantification of proteins by means of LC-MS/MS and the transition from targeted to untargeted omics approaches relying on pattern recognition/pattern discrimination as a key technology for the establishment of diagnostic decisions.

Toxicological oral fluid results among Spanish drivers testing positive on on-site drug controls from 2013 to 2015.

BACKGROUND: Driving under the influence of drugs (DUID) increases the risk of serious injury or death in traffic accidents. The aim of this study was to provide information about DUID in Spanish drivers. METHODS: 10,064 oral fluid samples were collected from Spanish drivers that tested positive on the roadside using the Dräger DrugTest 5000 (DDT5000) between 2013 and 2015. Samples were collected using Quantisal™ and analysed by LC-MS/MS at the Toxicology Laboratory of the Institute of Forensic Science of the University of Santiago de Compostela. RESULTS: Drivers were mainly young men (85.1% male, 29.7 ± 8.1 years old). In 98.5% of cases, LC-MS/MS results confirmed at least one of the positive results detected on the roadside. Cannabis (82.4%) and cocaine (42.1%) were the most commonly detected drugs. Poly-drug use was observed in 42.7% of drivers, mostly for all illicit drugs (>80%) except for cannabis (42.6%). Illicit drug and single-drug use was more frequent among drivers under 35 years old, and medicines and poly-drug use more common among drivers older than 35 years old. The on-site device performance was calculated using both the DDT5000 cut-offs and the LC-MS/MS method LOQs. Sensitivity (>73% vs >58%), specificity [>94% for all the compounds regardless the cut-offs used, except for cannabis (71%)] and accuracy (>87.5% with both cut-offs) fulfilled the DRUID Project requirements in all cases. CONCLUSION: LC-MS/MS confirmation result was negative in only 1.5% of the cases. The DUID driver profile was a young man, consuming cannabis or a combination of cannabis and cocaine.

Development, validation and application of a novel HPLC-MS/MS method for the quantification of atorvastatin, bisoprolol and clopidogrel in a large cardiovascular patient cohort.

Cardiovascular disease is a leading cause of morbidity, mortality, and healthcare expenditure worldwide. Importantly, there is interindividual variation in response to cardiovascular medications, leading to variable efficacy and adverse events. Therefore a rapid, selective, sensitive and reproducible multi-analyte HPLC-MS/MS assay for the quantification in human plasma of atorvastatin, its major metabolites 2-hydroxyatorvastatin, atorvastatin lactone and 2-hydroxyatorvastatin lactone, plus bisoprolol and clopidogrel-carboxylic acid has been developed, fully validated, and applied to a large patient study. Fifty microliter plasma samples were extracted with a simple protein precipitation procedure involving acetonitrile with acetic acid (0.1%, v/v). Chromatographic separation was via a 2.7 µm Halo C18 (50 × 2.1 mm ID, 90 Å) column and gradient elution at a flow rate of 500 µL/min consisting of a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid (v/v), over a 6.0 min run time. The six analytes and their corresponding six deuterated internal standards underwent positive ion electrospray ionisation and were detected with multiple reaction monitoring. The developed method was fully validated with acceptable selectivity, carryover, dilution integrity, and within-run and between-run accuracy and precision. Mean extraction recovery for the analytes was 92.7-108.5%, and internal standard-normalised matrix effects had acceptable precision (coefficients of variation 2.2-12.3%). Moreover, all analytes were stable under the tested conditions. Atorvastatin lactone to acid interconversion was assessed and recommendations for its minimisation are made. The validated assay was successfully applied to analyse 1279 samples from 1024 patients recruited to a cardiovascular secondary prevention prospective study.

Mass spectrometry approaches to metabolic profiling of microbial communities within the human gastrointestinal tract.

The interaction between microbial communities and their environment, such as the human gastrointestinal tract, has been an area of microbiology rapidly advanced, by developments in sequencing technology. However, these techniques are largely limited to the detection of the taxonomic composition of a microbial community and/or its genetic functional capacity. Here, we discuss a range of mass spectrometry-based approaches which researchers can employ to explore the host-microbiome interactions at the metabolic level. Traditional approaches to mass spectrometry are detailed, alongside new developments in the field, namely ambient ionisation mass spectrometry and imaging mass spectrometry, which we believe will prove to be important to future work in this field. We further discuss considerations for experimental workflows, data analysis options and propose a methodology for the establishment of causal relationships between functional host-microbiome interactions with regards to health and disease in the human gastrointestinal tract.

Development of a simultaneous LC-MS/MS method to predict in vivo drug-drug interaction in mice.

Cocktail substrates are useful in investigating drug-drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC-MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0-240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUCratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC-MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.

Recent advances in analytical methods for the therapeutic drug monitoring of immunosuppressive drugs.

Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for the TDM of ISDs since 2011. The five most commonly prescribed immunosuppressive medications are critically reviewed: cyclosporine A, tacrolimus, sirolimus (rapamycin), everolimus, and mycophenolic acid. This review introduces the general background of TDM and ISDs and presents the recent developments in using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassays for the TDM of ISDs. Finally, a future perspective for these analytical methods is briefly discussed.

Development of new efficient method for isolation of phenolics from sea algae prior to their rapid resolution liquid chromatographic-tandem mass spectrometric determination.

The extraction of phenolic compounds from 4 different sea algae samples, three brown algae (Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen, Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase extraction using micro-elution solid-phase extraction (µ-SPE) plate method was studied. Prior to µ-SPE, 50mg of algae with 80% methanol mixture was extracted in hyphenated series by various extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in combination with ultrasound assisted extraction. The µ-SPE plate technique reduced the time of sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids), hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid resolution liquid chromatography coupled to mass spectrometry detection with negative ion electrospray ionization (RRLC-ESI-MS) using multiple reactions monitoring. LOQs of measured samples varied in the range 0.23-1.68ng/mL and LODs in the range 0.07-0.52ng/mL. The applied method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol, glycosides, and esters insoluble in methanol) in less than 5min (including alkaline or acidic hydrolysis of raw extracts) from sea algae extracts.

Development of a multi-residue method for the determination of human and veterinary pharmaceuticals and some of their metabolites in aqueous environmental matrices by SPE-UHPLC-MS/MS.

The aim of the present work was to develop and validate a multi-residue method for the analysis of 33 human and veterinary pharmaceuticals (non-steroidal anti-inflammatory drugs (NSAIDs)/analgesics, antibiotics and psychiatric drugs), including some of their metabolites, in several aqueous environmental matrices: drinking water, surface water and wastewaters. The method is based on solid phase extraction (SPE) followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and it was validated for different aqueous matrices, namely bottled water, tap water, seawater, river water and wastewaters, showing recoveries between 50% and 112% for the majority of the target analytes. The developed analytical methodology allowed method detection limits in the low nanograms per liter level. Method intra- and inter-day precision was under 8% and 11%, respectively, expressed as relative standard deviation. The developed method was applied to the analysis of drinking water (bottled and tap water), surface waters (seawater and river water) and wastewaters (wastewater treatment plant (WWTP) influent and effluent). Due to the selectivity and sensitivity of the optimized method, it was possible to detect pharmaceuticals in all the aqueous environmental matrices considered, including in bottled water at concentrations up to 31ngL-1 (salicylic acid). In general, non-steroidal anti-inflammatory drugs/analgesics was the therapeutic group most frequently detected, with the highest concentrations found in wastewaters (acetaminophen and the metabolite carboxyibuprofen at levels up to 615 and 120µgL-1, respectively).

Liquid Chromatography-Tandem Mass Spectrometry: An Emerging Technology in the Toxicology Laboratory.

In the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in routine toxicology laboratories. LC-MS/MS offers significant advantages over other traditional testing, such as immunoassay and gas chromatography-mass spectrometry methodologies. Major strengths of LC-MS/MS include improvement in specificity, flexibility, and sample throughput when compared with other technologies. Here, the basic principles of LC-MS/MS technology are reviewed, followed by advantages and disadvantages of this technology compared with other traditional techniques. In addition, toxicology applications of LC-MS/MS for simultaneous detection of large panels of analytes are presented.

New and Evolving Techniques for the Characterization of Peptide Therapeutics.

Advances in technologies related to the design and manufacture of therapeutic peptides have enabled researchers to overcome the biological and technological challenges that have limited their application in the past. As a result, peptides of increasing complexity have become progressively important against a variety of disease targets. Developing peptide drug products brings with it unique scientific challenges consistent with the unique physicochemical properties of peptide molecules. The identification of the proper characterization tools is required in order to develop peptide formulations with the appropriate stability, manufacturability, and bioperformance characteristics. This knowledge supports the build of critical quality attributes and, ultimately, regulatory specifications. The purpose of this review article is to provide an overview of the techniques that are employed for analytical characterization of peptide drug products. The techniques covered are highlighted in the context of peptide drug product understanding and include chemical and biophysical approaches. Emphasis is placed on summarizing the recent literature experience in the field. Finally, the authors provide regulatory perspective on these characterization approaches and discuss some potential areas for further research in the field.

Development and validation of a HPLC method for determination of degree of polymerization of xylo-oligosaccharides.

A reliable reversed-phase HPLC method was developed for high resolution separation and high sensitivity determination of xylo-oligosaccharides (XOS) with degree of polymerization from 2 to 8. The method was carried out on a Kromasil C18 column using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and UV detection at 245nm. The effects of pH value of mobile phase, volume proportion of acetonitrile, concentration of ammonium acetate buffer and flow rate on the retention time and degree of separation of XOS derivatives were investigated. A satisfactory result was achieved in 25min with a mobile phase of 10mmol/L ammonium acetate buffer (pH5.5)-acetonitrile by a gradient elution at 0.8mL/min. In addition, this method was validated by liquid chromatography-tandem mass spectroscopy (LC-MS) analysis and several uncertain compounds were identified. The proposed HPLC method is suitable for the compositional analysis and quality control of XOS.

Biosampling strategies for emerging drugs of abuse: towards the future of toxicological and forensic analysis.

The term "new psychoactive substances" refers to emerging drugs of abuse whose chemical structure and psychoactive effects are similar to other already known compounds, often providing a "legal" alternative to internationally regulated drugs and mostly available via on-line retail sites. There are several categories of new psychoactive substances, such as synthetic cannabinoids, cathinone analogues, phenethylamines, tryptamines, and the need to identify and quantify an unprecedented and growing number of new compounds represents a unique challenge for toxicological and forensic analysis. The purpose of this review is to highlight biosampling, sample preparation and analysis of the most important classes of emerging drugs of abuse in biological matrices, focusing on alternatives to classical blood and urine "in tube" approach, still representing the standard routine for bioanalysis, despite inherent flaws regarding handling, stability and process feasibility. Chromatographic techniques coupled to mass spectrometry are usually exploited to identify and quantify new psychoactive substances; due to their high sensitivity and selectivity, it is possible to determine low concentrations not only in plasma and urine, but also in alternative matrices like dried blood spots, oral fluid, hair, other body fluids and tissues. Current literature on analytical methodologies applied to these samples is still limited and a more thorough validation is often required, including a comparison among the results obtained from conventional approaches and from innovative strategies, in order to determine their actual suitability.

Recent developments in the chromatographic bioanalysis of approved kinase inhibitor drugs in oncology.

In recent years (2010-present) there has been an increase in the number of publications reporting the development, validation and use of bioanalytical methods in the rapidly expanding drug class of small molecule protein kinase inhibitors. Most reports describe the technological set-up of the methods that have allowed for drug concentration measurements from various sample types. This includes plasma, dried blood-spot, and tissue-analysis. Also method development, exploration of various techniques, as well as measurement and identification of metabolites were addressed. For the bioanalysis, a variety of sample-pretreatment methods like protein-precipitation, liquid-liquid extraction, and solid-phase extraction have been employed, all varying in complexity, cleanliness and time-consumption. Chromatographic separation, nowadays, is more focused on separating components from ion-suppressive effects, since for MS/MS detection, various components do not have to be baseline separated. For detection multiple types of detectors were used, ranging from state-of-the-art high resolution, and tandem mass spectrometry with low picogram per milliliter detection limits to the classical UV-detector with several nanograms per milliliter limits. As new bioanalytical methods have arisen that do rely on chromatographic separation, for example for high-throughput analysis, these are addressed in this review as well.

[Mass spectrometry: past and present]. / Tömegspektrometria: múlt és jelen.

Mass spectrometry is a highly sensitive high-throughput instrumental analytical technique. It is used to determine the molecular mass, but also gives information on molecular structure amd is used for quantitation as well. Although it was developed over 100 years ago, it continues to evolve, both with respect to figures of merit (like sensitivity) and with respect to applications in various novel fields of science and technology. Mass spectrometry is capable of studying macromolecules (like proteins and protein complexes), and has very high sensitivity, now compounds at the atto- or zeptomol level can also be studied. Mass spectrometry can be coupled to separation techniques, and can be used to analyze complex mixtures, trace level compounds in biological matrices like active pharmaceutical ingredients or metabolites. In recent years in proteomics research has become a major new direction. In the present review we briefly introduce basic mass spectrometry techniques (ion surces, analyzers), combinations with chromatography (GC/MS, HPLC/MS), CEI MS) and tandem mass spectrometry. We also introduce two novel methods, mass spectrometry "imaging" and "lab-on-a-chip" technology.

LC-MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study.

A simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC-MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50mm×2.1mm, 2.6µm) with acteonitrile: 10mM ammonium formate buffer (80:20v/v) as an isocratic mobile phase with a flow rate of 0.5mLmin(-1). Quantitation was performed by transition of 264.1→151.1 (m/z) for busulfan and 272.1→159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2ngmL(-1) with a 100µL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100ngmL(-1) (r(2)≥0.9986). Chromatographic separation was achieved within 2.0min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration.