Development of a new ultra-high-performance liquid chromatography–tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC–MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid–liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC–MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.     

Development of a fast liquid chromatography–tandem mass spectrometry method for the determination of endocrine-disrupting compounds in waters

A fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed to study five endocrine-disrupting compounds (4-n-nonylphenol, bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol) in water. Different columns were tested; the chromatographic separation of the analytes was optimized on a Pinnacle DB biphenylic column with a water–acetonitrile gradient elution, which allowed the separation of the selected endocrine-disrupting compounds (EDCs) in less than 6 min. Quantitative analysis was performed in selected reaction monitoring (SRM) mode; two transitions were chosen for each compound, using the most abundant for quantitation. Calibration curves using bisphenol A-d ₁₆ as internal standard were drawn, showing good correlation coefficients (0.9993–0.9998). All figures of merit of the method were satisfactory; limits of detection were in the low pg range for all analytes. The method was then applied to the determination of the analytes in real water samples: to this aim, polar organic chemical integrative samplers (POCIS) were deployed in the influent and in the effluent of a drinking water treatment plant in Liguria (Italy). The EDC level was rather low in the influent and negligible in the outlet, reflecting the expected function of the treatment plant.     

Simultaneous quantitative determination of liraglutide and insulin degludec in rat plasma by liquid chromatography–tandem mass spectrometry method and its application

A simple, fast and high-throughput LC–tandem mass spectrometry method was developed and validated to simultaneously measure liraglutide and insulin degludec in rat plasma. After protein precipitation, plasma samples were subjected to gradient elution using an InertSustain Bio C₁₈ column with 1000/20/1 water/acetonitrile/formic acid (v/v/v) and 1000/1 acetonitrile/formic acid (v/v) as the mobile phase. The method was validated from 1.00 to 500 ng/mL of liraglutide and insulin degludec. Further, the extraction recovery from the plasma was 41.8%–49.2% for liraglutide and 56.5%–69.7% for insulin degludec. Intra- and inter-day precision of liraglutide was 3.5%–9.4% and 8.4%–9.8%, respectively, whereas its accuracy was between −12.6% and −1.3%. Intra- and inter-day precision of insulin degludec was 5.2%–13.6% and 11.8%–19.1%, respectively, showing an accuracy between −3.0% and 9.9%. As a result, the method was successfully applied to a pharmacokinetics study of liraglutide and insulin degludec following a single-dose subcutaneous administration to rats.     

Ultra-high-pressure liquid chromatography–tandem mass spectrometry method for the determination of alkylphenols in soil

A novel method has been developed for the determination of alkylphenols in soil by ultra-high-pressure liquid chromatography employing small particle sizes, combined with tandem mass spectrometry. Soil samples were extracted with pressurized liquid extraction (PLE) and then cleaned with solid-phase extraction (SPE). The extracts were separated on C18 column (1.7 μm, 50 mm × 2.1 mm) with a gradient elution and a mobile phase consisting of water and acetonitrile, and then detected by an electrospray ionization tandem mass spectrometry in negative ion mode with multiple reaction monitoring (MRM). Compared with traditional liquid chromatography, it took ultra-high-pressure liquid chromatography much less time to analyze alkylphenols. Additionally, the ultra-high-pressure liquid chromatography/tandem mass spectrometry method produces satisfactory reliability, sensitivity, and accuracy. The average recoveries of the three target analytes were 74.0–103.4%, with the RSD < 15%. The calibration curves for alkylphenols were linear within the range of 0.01–0.4 μg/ml, with the correlation coefficients greater than 0.99. When 10 g soil sample was used for analysis, the limits of quantification (LOQs) of the three alkylphenols were all 1.0 μg/kg.     

Development and validation of a pressurized liquid extraction liquid chromatography–tandem mass spectrometry method for perfluorinated compounds determination in fish

This paper describes the development and validation of an analytical methodology to determine eight perfluorinated compounds (PFCs) in edible fish using pressurized liquid extraction (PLE) with water and solid-phase extraction (SPE) with an ion-exchanger as extraction and pre-concentration procedures, followed by liquid chromatography–quadrupole-linear ion trap mass spectrometry (LC–QqLIT–MS). The rapidity and effectiveness of the proposed extraction procedure were compared with those most commonly used to isolate PFCs from fish (ion-pairing and alkaline digestion). The average recoveries of the different fish samples, spiked with the eight PFCs at three levels (the LOQ, 10 and 100 μg kg⁻¹ of each PFC), were always higher than 85% with relative standard deviation (RSD) lower than 17%. A good linearity was established for the eight PFCs in the range from 0.003–0.05 to 100 μg kg⁻¹, with r > 0.9994. The limits of quantification (LOQs) were between 0.003 and 0.05 μg kg⁻¹, which are well below those previously reported for this type of samples. Compared with previous methods, sample preparation time and/or LOQs are reduced. The method demonstrated its successful application for the analysis of different parts of several fish species. Most of the samples tested positive, mainly for perfluoropentanoic acid (PFPA), perfluorobutane sulfonate (PFBS) and perfluorooctanoic acid (PFOA) but other of the eight studied PFCs were also present.     

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma

A new, sensitive and fast method for the simultaneous determination of pyrazinamide, isoniazid, streptomycin, ethambutol, and rifampicin in human plasma was developed and validated. The method required only 100 μL of plasma and one step for sample preparation by protein precipitation. The drugs were separated by using a hydrophilic interaction liquid chromatography (HILIC) column. The mobile phase was methanol and water (0.1 % formic acid and 5 mM ammonium acetate, pH 3.0?±?0.1) in a ratio of 65:35 (v/v), which was eluted at an isocratic flow rate of 0.5 mL/min. Tandem mass spectrometry was performed with a triple-quadrupole tandem mass spectrometer. By use of the HILIC column, the detection was free of ion-pair reagents in the mobile phase, with no significant matrix effects. The total run time was less than 2 min for each sample. The method was validated by evaluating its selectivity, sensitivity, linearity, accuracy, and precision according to US Food and Drug Administration guidelines. The lower limit of quantification was 4.0 ng/mL for pyrazinamide, isoniazid, and rifampicin, 0.5 ng/mL for ethambutol, and 10.0 ng/mL for streptomycin. The intraday precision and interday precision were less than 9 %, with the accuracy ranging between ?9.3 and 7.3 %. The method was successfully applied to therapeutic drug monitoring of 33 patients with tuberculosis after administration of standard antituberculosis drugs. The method has been proved to meet the high-throughput requirements in therapeutic drug monitoring.

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantitation of microcystin-RR and its metabolites in fish liver

A novel method for identification and quantification of microcystin-RR (MC-RR) and its metabolites (MC-RR-GSH and MC-RR-Cys) in the fish liver was developed and validated. These analytes were simultaneously extracted from fish liver using water containing EDTA with 5% acetic acid, followed by a mixed-mode cation-exchange SPE (Oasis MCX) and subsequently determined by liquid chromatography–electrospray ionization ion trap mass spectrometry (LC–ESI-ITMS). Extraction parameters including volume and pH of eluting solvents, were optimized. Best recoveries were obtained by using 10 mL of 15% ammonia solution in methanol. The mean recoveries at three concentrations (0.2, 1.0, and 5.0 μg g⁻¹ dry weight [DW]) for MC-RR, MC-RR-GSH and MC-RR-Cys were 93.6–99%, 68.1–73.6% and 90.0–95.2%, respectively. Method detection limit (MDL) were 4, 7 and 5 ng g⁻¹ DW for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. Limits of quantification (LOQs) for MC-RR, MC-RR-GSH and MC-RR-Cys were calculated to be 10, 18 and 13 ng g⁻¹ DW, respectively. Finally, this method was successfully applied to the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in the liver of bighead carp with acute exposure of MCs.     

Determination of meloxicam in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to detect meloxicam in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source was used in positive ion mode. Protein precipitation with acetonitrile was used for sample preparation. Meloxicam and ¹³C₆-meloxicam internal standard were analyzed on an Acquity CSH C₁₈ column with a mobile phase of acetonitrile and water in 0.1% formic acid using a gradient program for separation. The retention time of meloxicam was 1.1 min and the total run time was only 2.0 min. Detection was performed in multiple reaction monitoring mode using an electrospray ionization source with optimized mass spectrometry parameters. The calibration curves were linear in the range 10.0–3.00 × 10³ ng/ml (r ≥ 0.99). The within-run and between-run RSDs were ≤14.8%. The within-run and between-run REs ranged from −4.6 to 10.7%. There was no significant matrix effect, and the recovery rate was high. This method was fully validated, including reinjection reproducibility in human plasma. The method was applied to the pharmacokinetic study. All of the incurred sample reanalysis methods met the criteria.     

Detection of catecholamine metabolites in urine based on ultra-high-performance liquid chromatography–tandem mass spectrometry

The excretion of neurotransmitter metabolites in normal individuals is of great significance for health monitoring. A rapid quantitative method was developed with ultra-performance liquid chromatography–tandem mass spectrometry. The method was further applied to determine catecholamine metabolites vanilymandelic acid (VMA), methoxy hydroxyphenyl glycol (MHPG), dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) in the urine. The urine was collected from six healthy volunteers (20–22 years old) for 10 consecutive days. It was precolumn derivatized with dansyl chloride. Subsequently, the sample was analyzed using triple quadrupole mass spectrometry with an electrospray ion in positive and multireaction monitoring modes. The method was sensitive and repeatable with the recoveries 92.7–104.30%, limits of detection (LODs) 0.01–0.05 μg/mL, and coefficients no less than 0.9938. The excretion content of four target compounds in random urine samples was 0.20 ± 0.086 μg/mL (MHPG), 1.27 ± 1.24 μg/mL (VMA), 3.29 ± 1.36 μg/mL (HVA), and 1.13 ± 1.07 μg/mL (DOPAC). In the urine, the content of VMA, the metabolite of norepinephrine and adrenaline, was more than MHPG, and the content of HVA, the metabolite of dopamine, was more than DOPAC. This paper detected the levels of catecholamine metabolites and summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.     

Optimization of a method for the determination of a mustard gas biomarker in human blood plasma by liquid chromatography–high-resolution mass spectrometry

A method for the determination of a mustard gas biomarker (an S-hydroxyethylthioethyl adduct with albumin) in blood plasma was optimized with the use of HPLC with high-resolution tandem mass-spectrometric detection. This method is based on the hydrolysis of this adduct by the proteinase K enzyme with the formation of the following stable tripeptide with cysteine, proline, and phenylalanine: S-[HETE]-Cys- Pro-Phe. The conditions of the sample preparation of human plasma artificially contaminated by mustard gas (the selection of an aliquot portion volume and an enzyme for the hydrolysis), the mass-spectrometric detection (the selection of optimum pairs of ion reactions and high-resolution detection modes), and the gradient elution program in the HPLC separation of an analyzed mixture were optimized. The detection limit of mustard gas in human blood plasma was 1 ng/mL. The approach proposed was tested in the analysis of human blood plasma samples by the standard addition technique and also within the framework of the first official biomedical test carried out by the Organization for the Prohibition of Chemical Weapons (OPCW) in 2016, and it exhibited a good accuracy, reproducibility, and specificity of determination.     

In-house validation and factorial effect analysis of a liquid chromatography–tandem mass spectrometry method for the determination of thyreostats in bovine blood plasma

A sensitive and robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method allowing the rapid screening and confirmation of thyreostatic drugs in bovine blood plasma was developed and validated according to Commission Decision 2002/657/EC, chapter 3.1.3 “alternative validation”, by applying a matrix-comprehensive in-house validation concept. Decision limit CCα, detection capability CCβ, recovery, repeatability, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and duration of sample preparation) were systematically varied on two levels during the validation study. Subsequently, the extent to which these factors influence the measurement results of the individual analytes was examined.     

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Sensitive liquid chromatography–tandem mass spectrometry method for the determination of pantoprazole sodium in human urine

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed to determine pantoprazole sodium (PNT) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C₁₈ column (symmetry 3.5 μm; 75 mm × 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (90:10, v/v). The method was linear over a concentration range of 1–100 ng mL^?1. The lower limit of quantitation was 1 ng mL^?1. The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was <10.5%. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ng mL^?1 PNT) was within ±1.25% in terms of relative errors.     

Development and validation of a multi-residue method for the determination of pesticides in processed fruits and vegetables using liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive multi-residue analytical method, utilizing ethyl acetate extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), has been developed and validated for simultaneous determination of 28 pesticides of different chemical classes (polar organophosphates, carbamates, strobilurines, neonicotinoids, amides, pyrimidines, benzimidazoles, imidazoles and triazoles), and their transformation products, in processed fruit and vegetables. Two precursor-product ion transitions were monitored for each pesticide in selected reaction monitoring (SRM) mode. Linearity (r (2) > or = 0.99) was good over the concentration range 0.5 to 100 microg L(-1) for all the pesticides, and instrumental detection limits ranged from 0.1 to 1 microg L(-1). Mean recovery for fruit and vegetables spiked at 0.010 mg kg(-1) ranged from 65 to 94.4%, and relative standard deviations ranged from 9.0 to 20.0%. When the amount spiked was 0.050 mg kg(-1) recoveries ranged from 72.5 to 90% and relative standard deviations were from 6.1 to 19.0%. Method detection limits were from 0.002 to 0.007 mg kg(-1) for the different food matrices studied. The method was used to monitor pesticide residues in a wide variety of fruits and vegetables.     

Solid-phase extraction liquid chromatography–tandem mass spectrometry analytical method for the determination of 2-hydroxy-4-methoxybenzophenone and its metabolites in both human urine and semen

2-Hydroxy-4-methoxybenzophenone (HMB), which is one of the most commonly used UV filters in sunscreen cosmetics to protect skin from the deleterious effects of the sun, can be percutaneously absorbed, further metabolized, and finally excreted or bioaccumulated. An analytical method for the sensitive determination of HMB and its three metabolites in both human urine and semen is developed. The presented analytical method is based on a solid-phase extraction (SPE) procedure to clean-up and preconcentrate the target analytes from the urine and semen samples followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection. The methodology was fully validated and the standard addition calibration method was used to quantify the target analytes in order to correct the matrix effects observed. Considering this approach, the accuracy of the method was evaluated and the recoveries ranged from 98% to 115% and from 86% to 111% in urine and semen samples, respectively, depending on the analyte. For urine samples, the limits of detection ranged between 0.027 and 0.103 ng mL⁻¹ and the repeatability of the method, expressed as relative standard deviation, was in the range of 7.2–9.2%, depending on the analyte. In the case of semen samples, the limits of detection ranged between 1 and 3 ng mL⁻¹ whereas the repeatability was in the range of 2.2–6.4%, depending on the analyte. The described SPE-LC-MS/MS method was satisfactorily applied to both urine and semen samples from a male volunteer who applied a sunscreen cosmetic product containing HMB. HMB and its metabolites were found and quantified in the low ng mL⁻¹ range in both urine and semen samples, although at a different extent.     

Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatographic–tandem mass spectrometric (HILIC–MS–MS) method for investigation of the in vivo metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen, in rabbit blood has been developed and validated. This method achieved excellent repeatability and accuracy. Recovery ranged from 76.9 to 116.3 % and precision (as RSD) between 0.53 and 6.52 %. Linearity was good for all compounds (R ²?>?0.9990) and the limit of detection (LOD) ranged from 0.016 to 0.082 ng mL^?1. Pharmacokinetic analysis indicated that NNK was rapidly eliminated in vivo in rabbit blood and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite. The hydroxy acid, keto acid, and NNAL-N-oxide were also important metabolites in rabbit blood. It is probable that α-methylene hydroxylation was the major pathway of α-hydroxylation of NNK and NNAL in the rabbit.

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]⁺ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]^- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS ⁿ analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.