Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry for Therapeutic Drug Monitoring of Metformin and Rosuvastatin in Human Plasma

In this work a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) has been developed and fully validated for the quantitation of metformin and rosuvastatin in human plasma. Sample preparation involved the use of 100 µL of human plasma, following protein precipitation and filtration. Metformin, rosuvastatin and 4-[2-(propylamino) ethyl] indoline 2 one hydrochloride (internal standard) were separated by using an X-Bridge-HILIC BEH analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm) with isocratic elution. A mobile phase consisting of 12% (v/v) 15 mM ammonium formate water solution in acetonitrile was used for the separation and pumped at a flow rate of 0.25 mL min−1 . The linear range of the assay was 100 to 5000 ng mL−1 and 2 to 100 ng mL−1 for metformin and rosuvastatin, respectively. The current HILIC-ESI/MS method allows for the accurate and precise quantitation of metformin and rosuvastatin in human plasma with a simple sample preparation and a short a chromatographic run time (less than 15 min). Plasma samples from eight patients were further analysed proving the capability of the proposed method to support a wide range of clinical studies

Ultra-Fast Retroactive Processing by MetAlign of Liquid-Chromatography High-Resolution Full-Scan Orbitrap Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program

Rationale: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. Methods: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). Results: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. Conclusions: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.</p

Population Reference Ranges of Urinary Endogenous Sulfate Steroids Concentrations and Ratios as Complement to the Steroid Profile in Sports Antidoping

The population based Steroid Profile (SP) ratio of testosterone (T) and epitestosterone (E) has been considered as a biomarker approach to detect testosterone abuse in '80s. The contemporary Antidoping Laboratories apply the World Antidoping Agency (WADA) Technical Document (TD) for Endogenous Androgenic Anabolic Steroids (EAAS) in the analysis of SP during their screening. The SP Athlete Biological Passport (ABP) adaptive model uses the concentrations of the total of free and glucuronide conjugated forms of six EAASs concentrations and ratios measured by GC/MS. In the Antidoping Lab Qatar (ADLQ), the routine LC/MS screening method was used to quantitatively estimate the sulfate conjugated EAAS in the same analytical run as for the rest qualitative analytes. Seven sulfate EAAS were quantified for a number of routine antidoping male and female urine samples during screening. Concentrations, statistical parameters and selected ratios for the 6 EAAS, the 6 sulfate EAAS and 29 proposed ratios of concentrations from both EAAS and sulfate EAAS, which potentially used as SP ABP biomarkers, population reference limits and distributions have been estimated after the GC/MSMS analysis for EAAS and LC/Orbitrap/MS analysis for sulfate EAAS

Application of liquid chromatography / time of flight mass spectrometry for the doping control of prohibited substances in athlete's and horse urine

In the current study, new qualitative and quantitative methods were developed andvalidated for the doping control screening analysis, confirmation, and/or quantitativedetermination of prohibited substances using LC/TOF-MS technology. The first part of thiswork presents the use of LC/TOF-MS for the multiple detection of a great number ofprohibited substances in athletes urine. In the second part, the same technology was appliedfor the quantification and identification of 4 threshold substances in horse urine using directinjection analysis.Unification of screening protocols for a wide range of doping agents has become animportant issue for doping control laboratories. In the present study, a high throughputscreening method was developed for the multiple detection of 266 small molecule analytesfrom all categories of prohibited substances included in the WADA List. The proposedmethodology is based on a single-step liquid-liquid extraction of hydrolyzed urine and theuse of a rapid-resolution LC/TOF-MS system acquiring continuous full scan spectral data.Electrospray ionization in the positive mode was applied. The extraction protocol andsample preparation procedure used in this study was applied in the OACA doping controllaboratory for the routine screening analysis of anabolic agents and corticosteroids. Ourapproach on sample preparation was to study the efficiency of this procedure for otherclasses of substances (stimulants, narcotics, diuretics). Validation parameters consisted ofidentification capability, limit of detection, specificity, ion suppression, extraction recovery,repeatability and mass accuracy. Detection criteria were established on the basis ofretention time reproducibility and mass accuracy. The suitability of the methodology fordoping control was demonstrated with positive urine samples. The preventive role of themethod was proved by the case where full scan acquisition with accurate massmeasurement allowed the retrospective reprocessing of acquired data from past dopingcontrol samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine,which resulted in re-reporting a number of stored samples as positives for this particularsubstance, when, initially, they had been reported as negatives.Hydrocortisone is the primary endogenous glucocorticosteroid and is used in equineveterinary medicine for its anti-inflammatory properties. As an endogenous substance, itscontrol in equine sports is regulated by a threshold value of 1 μg ml-1 free hydrocortisone inurine. Two simple and rapid LC/MS methods with direct injection analysis were developedand validated for the quantification and identification of hydrocortisone in equine urineusing the same sample preparation but different mass spectrometric systems: IT-MS andTOF-MS. The main advantage of the proposed methodology is the minimal samplepreparation procedure, as particle-free diluted urine samples were directly injected intoboth LC/MS systems. Desonide was used as internal standard (IS). The tested linear rangewas 0,25–2,5 μg ml−1 for both methods. Matrix effects were evaluated by preparing andanalyzing calibration curves in water solutions and different horse urine samples. A greatvariation of the signal both for hydrocortisone and the internal standard was observed indifferent matrices. To overcome matrix effects, the unavailability of blank matrix and theexcessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standardadditions approach in a method where neither non isotopic internal standards nor extensivesample preparation is utilized and no blank matrix is available. Validation results on linearity,accuracy and precision for both methods prove their suitability for the doping controlanalysis of hydrocortisone in horse urine. The above method was applied to a real sample,using both LC/MS methods.In equine sport, theobromine is prohibited with a threshold level of 2 μg mL-1 in urine. Twosimple LC/MS methods for the identification andquantification of theobromine weredeveloped and validated using the same sample preparation procedure but different massspectrometric systems (IT-MS and TOF-MS. Particle-free diluted urine samples were directlyinjected into the LC/MS systems, avoiding the time-consuming extraction step. 3-Propylxanthine was used as the internal standard. The tested linear range was 0.75–15 μgmL-1. Matrix effects were evaluated analyzing calibration curves in water and differentfortified horse urine samples. A great variation in the signal of theobromine and the internalstandard was observed in different matrices. To overcome matrix effects, a standardadditions calibration method was applied. The linearity, accuracy and precision for bothmethods prove their suitability for the doping control analysis of theobromine in horseurine. The methods were applied to two case samples, demonstrating simplicity, accuracyand selectivity.Salicylates have analgesic, anti-inflammatory, and antipyretic properties. In horses,salicylates may derive both from feeding as well as from medication. In equine sport,salicylic acid is prohibited with a threshold level of 750 μg mL−1 in urine. A simple and rapidLC/MS method was developed and validated for the quantification and identification ofsalicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the LC/QTOF-MS system. Electrosprayionization in negative mode with full scan acquisition mode and product ion scan mode werechosen for the quantification and identification of salicylic acid, respectively. Run time was2.0 min. The tested linear range was 2.5–50 μg mL−1 (after 100-fold sample dilution). Thelinearity, accuracy, and precision results prove the method’s suitability for the dopingcontrol analysis of salicylic acid in horse urine. The area ratios of the diagnostic product ionsof salicylic acid were found to be precise for confirmation purposes. The above method wasapplied to the quantitative and confirmatory analysis of two real samples.The endogenous catecholamine dopamine and its pharmacologically inert precursor, L-dopa,are both available as human pharmaceutical preparations and can be used as performanceenhancing drugs in horses. Urinary 3-methoxytyramine is used as an indicator ofdopaminergic manipulation resulting from dopamine or levodopa administration and isprohibited with a urinary threshold of 4 μg mL−1 (free and conjugated). A simple LC/MSmethod was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis andprotein precipitation. ΗILIC was selected as a separation technique that allows effectiveretention of polar substances like 3-methoxytyramine and efficient separation from matrixcompounds. Electrospray ionization (ESI) in positive mode with product ion scan mode waschosen for the detection of the analytes. Studies on matrix effects showed ion suppressiondepending on the horse urine used. To overcome the variability of the results originatingfrom the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. Thelinearity, accuracy and precision results prove the method’s suitability for the doping controlanalysis of 3-methoxytyramine in horse urine. The above method was applied to thequantitative and confirmatory analysis of three case samples

High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid–liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC–MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files’ reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis

Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine

Rationale: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. Methods: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data. Results: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. Conclusions: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network.</p