Development of rapid and high‐throughput LC–MS/MS method for quantification of olprinone in rabbit plasma

A simple, highly sensitive and rapid method for quantification of olprinone (phosphodiesterase 3 inhibitor) in rabbit plasma using liquid chromatography–tandem mass spectrometry with electrospray was developed. An aliquot of 50 μL of plasma sample was cleaned up and extracted using Ostro? 96‐well plate followed by dilution. Chromatographic separation of olprinone and olprinone‐d3 was carried out on a CORTECS^® T3 column within 3 min. Detection was achieved using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive ion multiple reaction monitoring mode using the transitions m/z 251.07 → m/z 155.06 and m/z 254.21 → m/z 158.10 for olprinone and olprinone‐d3, respectively. The method was validated according to US Food and Drug Administration guideline for bioanalytical methods, and showed excellent linearity in the range 10.0–2000.0 ng/mL with coefficient of determination >0.99. The intra‐ and inter‐day precisions (CV) were <5.1% and the accuracies were within the range 99.7–103.2% at all quality control concentrations. Furthermore, olprinone was stable under various stability conditions. The developed method was used for quantification of olprinone in rabbit plasma after its intravenous administration at the dose of 1 mg/kg in order to better understand the metabolism of olprinone in a rabbit model of lung injury.     

Orbitrap™ monostage MS versus hybrid linear ion trap MS: application to multi‐allergen screening in wine

Food allergen research has made giant steps in the last years thanks to the features offered by the latest technology of mass analyzers placed on the market allowing multiplex sensitive detection of proteins. Potentials and features of two mass analyzers namely a linear ion trap capable of performing a data dependent or selected reaction monitoring analysis and an Orbitrap^TM stand‐alone MS enabling a broadband fragmentation without mass selection at highest mass resolving power are herein described and applied to the multiplex screening of allergens in a type of wine chosen as a reference matrix. Quantitative and confirmative capabilities of both platforms were assessed on the specific case study, the multiple detection of egg and milk ‐related proteins, typically employed in white wines as fining agents. Commercial bioinformatic tools used for a quick allergen identification will be also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

Identification of short‐chain poly‐3‐hydroxybutyrates in Saiga horn extracts using LC–MS/MS

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high‐performance liquid chromatography coupled with high‐resolution mass spectrometry, we have been able to identify a series of short‐chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high‐performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short‐chain poly‐3‐hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.     

Combined quantification of paclitaxel,docetaxel and ritonavir in human feces and urine using LC‐MS/MS

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 μL urine or 50 mg feces followed by high‐performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry‐over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel, 2–2000 ng/mL for ritonavir in urine, 2–2000 ng/mg for paclitaxel and docetaxel, and 8–8000 ng/mg for ritonavir in feces. Inter‐assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS

LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C₈ column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.     

Targeted profiling and relative quantification of benzoyl diterpene alkaloids in Aconitum roots by using LC–MS/MS with precursor ion scan

Benzoyl aconite alkaloids have myocardial protective effects at a low dose and produce toxic effects at high dose. Due to lack of enough reference compounds, most of the benzoyl alkaloids had few concerns, except the typical ones, i.e. aconitine, mesaconitine, and hypaconitine. To rapidly screen out and quantify benzoyl alkaloids, a high performance liquid chromatography combined with tandem mass spectrometry was proposed based on precursor ion scanning mode. First, a diagnostic ion at m/z 105 corresponding to benzoyl group was observed by using tandem mass spectrometry, which could be used for the rapid identification of benzoyl alkaloids. The targeted screening of these alkaloids was then conducted by using precursor ion scan of characteristic ion at m/z 105. Shengfuzi (the lateral root of A. carmichaelii) was taken as example, and 24 benzoyl‐containing alkaloids were identified. The six major alkaloids including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine were determined in the precursor ion scan mode by the standard curve method. Reliable linearity, sensitivity, precision, accuracy, and repeatability were obtained and validated. Then the relative response factors between these six analytes were calculated, which were not more than two times using any alkaloid as reference. Thus, the other 18 alkaloids lacking reference compounds were relatively quantified. This approach provides a useful tool for rapid identification and quantitative analysis of toxic benzoyl alkaloids, and also an efficient method for the safety assessment of Aconitum roots.     

Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed‐ and chiral‐phase LC–MS

LC with atmospheric pressure chemical ionization (ACPI) MS with RP and chiral phase was used for separation of triacylglycerols (TAGs) from yeasts of the genera Candida, Kluyveromyces, Rhodotorula, Saccharomyces, Torulospora, Trichosporon, and Yarrowia. Chiral LC–APCI‐MS is based on using two columns in series packed with a 3,5‐dimethylphenyl carbamate modified β‐cyclodextrin chiral phase. All regioisomers and enantiomers of TAGs containing one to five double bonds were separated. Molecular species of TAGs, i.e. regioisomers and enantiomers, were identified and quantified by MS/MS. Among the 94 identified TAGs, the most abundant were triolein, oleopalmitoleoolein, and dipalmitoleoolein. In strains producing palmitoleic acid in amounts >25% of total fatty acids (FAs), this acid, or unsaturated FA is bound in sn‐1. In strains containing palmitoleic acid at 10–25% total FAs this acid is mainly bound in sn‐3, saturated FA being bound in sn‐1. Strains containing <10% palmitoleic acid form preferentially symmetrical TAGs.     

Determination of avoparcin in animal tissues and milk using LC‐ESI‐MS/MS and tandem‐SPE

A highly sensitive and selective method using LC‐ESI‐MS/MS and tandem‐SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H]³⁺ and the major product ions of AV‐α and ‐β at m/z 637 → 86/113/130 and m/z 649 → 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem‐SPE with an ion‐exchange (SAX) and InertSep C18‐A cartridge clean‐up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV‐α (r >0.996) and ‐β (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).     

Fast ultrasound‐assisted extraction combined with LC–MS/MS of perfluorinated compounds in manure

A fast analytical method for the determination of perfluorinated compounds in poultry manure by LC–MS/MS was developed. The extraction was carried out by ultrasound‐assisted extraction of 1 g of sample, during 2 × 15 min using low volume (5.5 mL) of a mixture of methanol and acetonitrile. An efficient extraction of perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, and perfluoroalkyl sulfonamides from poultry manure was obtained with recoveries higher than 81%. The cleanup of extracts was carried out by dispersive SPE. The validation of the proposed method showed the suitability of this procedure to determine perfluorinated compounds in poultry manure with detection limits in the range of 0.44–2.12 ng/g, depending on the target compound. In comparison with previously published methods, the miniaturization of the sample preparation method with ultrasound‐assisted extraction together with the use of a core‐shell column permit a lower consumption of organic solvents and a fast analysis of perfluorinated compounds. Manure samples obtained from Spanish commercial farms were analyzed and low perfluorinated compounds levels were found, which may be originated by dietary or environmental exposure. The highest concentrations measured corresponded to the perfluoroalkyl sulfonates, which varied from 8.2 to 35.9 ng/g.     

Structural identification of degradants of moexipril by LC–MS/MS

A gradient LC–MS method was developed for the identification and characterization of degradants of moexipril using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). Moexipril was subjected to hydrolysis (acid, base and neutral), oxidation, photolytic and thermal degradation conditions as mentioned in ICH guidelines Q1A (R2). The drug degraded under hydrolysis, oxidation and photolytic conditions, but it was stable under thermal conditions. In total, five degradants were formed and separated on an Agilent XDB C‐18 column (4.6 × 150 mm, 5 μm) in a gradient elution method. Four degradants ( D1 , D2 , D4 and D5 ) under acidic conditions, three degradants ( D2 , D3 and D4 ) under basic conditions and three degradants ( D1 , D4 and D5 ) under neutral and oxidative stress conditions were formed. In addition, two degradants ( D4 and D5 ) were formed under photolytic stress conditions. To elucidate the structures of degradants, fragmentation of moexipril and its degradants was studied using LC–MS/MS experiments and accurate mass measurements (HRMS) data. The fragment ions in the product ion tandem mass spectra of all the degradants were compared with those of moexipril and assigned the probable structures for the degradants.     

High‐throughput LC–MS method for the rapid characterization of multiple chemical constituents and metabolites of Da‐Bu‐Yin‐Wan

Traditional Chinese medicine is the clinical experience accumulated by Chinese people against diseases. Da‐Bu‐Yin‐Wan is a famous traditional Chinese medicine formula consisting of Phellodendri amurensis Rupr., Anemarrhenae asphodeloides Bge., Radix Rehmanniae Preparata and Chinemys reevesii . In this study, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight high‐definition mass spectrometry with the control software of Masslynx (V4.1) was established for comprehensive screening and identification of the chemical constituents and serum metabolites of Da‐Bu‐Yin‐Wan in vivo and in vitro. Consequently, 70 peaks in the methanol extract from Da‐Bu‐Yin‐Wan and 38 peaks absorbed into rat blood were characterized. The 70 constituents in vitro included alkaloids, flavonoids, polysaccharide, limonoids, flavonoid, etc. And the 38 constituents consist of 22 absorbed prototypes and 16 metabolites of Da‐Bu‐Yin‐Wan absorbed in vivo. We fully clarified the chemical constituents of Da‐Bu‐Yin‐Wan and provided a scientific strategy for the screening and characterization of the chemical constituents and metabolites of traditional Chinese medicine in vitro and in vivo.     

Validated LC–MS/MS method for quantification of agomelatine in human plasma and its application in a pharmacokinetic study

An analytical method based on liquid–liquid extraction has been developed and validated for analysis of agomelatine in human plasma. Fluoxetine was used as an internal standard for agomelatine. A Betasil C18 (4.0 × 100 mm, 5 µm) column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatographic conditions and mass spectrometric detection in the positive ionization mode using an API‐4000 system. The proposed method has been validated with linear range of 0.050–8.000 ng/ml for agomelatine. The intra‐run and inter‐run precision values are within 12.12% and 9.01%, respectively, for agomelatine at the lower limit of quantification level. The overall recovery for agomelatine and fluoxetine was 67.10% and 72.96%, respectively. This validated method was used successfully for analysis of plasma samples from a pharmacokinetic study. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of LC–MS/MS method for quantification of bictegravir in human plasma and its application to an intracellular uptake study

A sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of bictegravir in human plasma. A small volume of only 50 μL aliquot of plasma was precipitated with acetonitrile containing an internal standard (IS). Chromatographic separation was performed on a Kinetex EVO C₁₈ column, 50 × 3.0 mm, 5 μm using an isocratic mobile phase containing 80:20 acetonitrile–water with 0.1% formic acid. A mass spectrometer was operated in ESI positive multiple reaction monitoring mode using the m/z 450.1/289.1 for bictegravir and 420.1/277.1 for IS. The dynamic range of the method was 1–10,000 ng/mL with a correlation coefficient of ≥0.9991. The precision results of calibration standards were in the range of 0.05–4.57% and accuracies were 95.07–104.70%. The mean extraction recovery was 98.64% with a precision of 2.91%. The method was validated as per US Food and Drug Administration guidelines and was found to be accurate and precise. The method was successfully applied to in vitro cellular uptake study.     

Detection and characterization of prednisolone metabolites in human urine by LC‐MS/MS

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β‐glucuronidase and subjected to liquid–liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β‐dihydro‐PRED and 20α‐dihydro‐PRED, 20β‐dihydro‐prednisone and 20α‐dihydro‐prednisone, 6β‐hydroxy‐PRED and 6α‐hydroxy‐PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21‐pentahydroxypregnan‐1,4‐diene‐3‐one, 6α,11β,17α,20β,21‐pentahydroxypregnan‐1,4‐diene‐3‐one and Δ⁶‐PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6‐hydroxy‐metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6). Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a single‐run analytical method for the detection of ten multiclass emerging contaminants in agricultural soil using an acetate‐buffered QuEChERS method coupled with LC–MS/MS

This study was undertaken to develop and validate a single multiresidue method for the monitoring of ten multiclass emerging contaminants, viz. ceftiofur, clopidol, florfenicol, monensin, salinomycin, sulfamethazine, sulfathiazole, sulfamethoxazole, tiamulin, and tylosin in agricultural soil. Samples were extracted using an acetate‐buffered, modified quick, easy, cheap, effective, rugged, and safe method followed by liquid chromatography with tandem mass spectrometric analysis in positive ion mode. Separation on an Eclipse Plus C₁₈ column was conducted in gradient elution mode using a mobile phase of methanol (A) and distilled water (B), each containing 0.1% formic acid and 5 mM ammonium formate. The linearity of the matrix‐matched calibrations, expressed as determination coefficients, was good, with R ² ≥ 0.9908. The limits of quantification were in the range 0.05–10 μg/kg. Blank soil samples spiked with 4 × and 20 × the limit of quantification provided recovery rates of 60.2–120.3% (except sulfamethoxazole spiked at 4 × the limit of quantification, which gave 131.9%) with a relative standard deviation < 13% (except clopidol spiked at 20 × the limit of quantification, which gave 25.2%). This method was successfully applied to the monitoring of 51 field‐incurred agricultural loamy‐sand soil samples collected from 17 provincial areas throughout the Korean Peninsula. The detected and quantified drugs were clopidol (≤ 4.8 μg/kg), sulfathiazole (≤ 7.7 μg/kg), sulfamethazine (≤ 6.6 μg/kg), tiamulin (≤ 10.0 μg/kg), and tylosin (≤ 5.3 μg/kg). The developed method is simple and versatile, and can be used to monitor various classes of veterinary drugs in soil.     

Determination of macrolide antibiotics residues in pork using molecularly imprinted dispersive solid‐phase extraction coupled with LC–MS/MS

A class‐specific macrolide molecularly imprinted polymer was synthesized by precipitation polymerization using tulathromycin as the template and methacrylic acid as the functional monomer. The polymers revealed different specific adsorption and imprinting factor for macrolides with different spatial arrangement of side chains as well as lactonic ring size. And the molecularly imprinted polymer possessed maximum adsorption capacity (54.1 mg/g) and highest imprinting factor (2.4) toward 15‐membered ring azithromycin. On the basis of molecularly imprinted polymer dispersive solid‐phase extraction, a rapid, selective, and reproducible method for simultaneous determination of seven macrolide antibiotics residues in pork was established by using liquid chromatography with tandem mass spectrometry. At spiking levels of 5, 10, 25, and 100 μg/kg, average recoveries of seven macrolides ranged from 68.6 to 95.5% with intraday and interday relative standard deviations below 8%. The limits of detection and limits of quantification were 0.2–0.5 and 0.5–2.0 μg/kg, respectively.     

LC‐MS analysis of estradiol in human serum and endometrial tissue: Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

Accurate measurement of estradiol (E2) is important in clinical diagnostics and research. High sensitivity methods are critical for specimens with E2 concentrations at low picomolar levels, such as serum of men, postmenopausal women and children. Achieving the required assay performance with LC–MS is challenging due to the non‐polar structure and low proton affinity of E2. Previous studies suggest that ionization has a major role for the performance of E2 measurement, but comparisons of different ionization techniques for the analysis of clinical samples are not available. In this study, female serum and endometrium tissue samples were used to compare electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) in both polarities. APPI was found to have the most potential for E2 analysis, with a quantification limit of 1 fmol on‐column. APCI and ESI could be employed in negative polarity, although being slightly less sensitive than APPI. In the presence of biological background, ESI was found to be highly susceptible to ion suppression, while APCI and APPI were largely unaffected by the sample matrix. Irrespective of the ionization technique, background interferences were observed when using the multiple reaction monitoring transitions commonly employed for E2 (m/z 271 > 159; m/z 255 > 145). These unidentified interferences were most severe in serum samples, varied in intensity between ionization techniques and required efficient chromatographic separation in order to achieve specificity for E2. Copyright © 2013 John Wiley & Sons, Ltd.