A validated method for the determination of traces of UV filters in fish using LC–MS/MS

An analytical method for the determination of UV filter substances in fish tissue has been developed and validated using benzophenone-3, 3-(4-methylbenzylidene)-camphor, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propenoate and 2-ethylhexyl 3-(methoxyphenyl)-2-propenoate as target analytes. The fish fillets were homogenised and extracted by Soxhlet extraction. The extracts were run through a clean-up process including gel permeation chromatography followed by solid-phase extraction. Quantification of the compounds was performed using liquid chromatography with tandem mass spectrometric detection. Blank fish as well as spiked blank fish were analysed to validate the analytical method. The analytical method developed has the multiple advantages of enabling separation, simultaneous identification and quantification of each of the four selected compounds in a single run. Contamination of blank samples and abnormally high concentrations in spiked samples were avoided by taking extensive precautions during the fish preparation procedure. The method was validated in accordance with internationally accepted criteria, such as specificity, accuracy and repeatability. The combination of LC with tandem mass spectrometry ensures a high level of specificity. The accuracy of the method was reported as the mean recovery rate for the analytes in the sample matrix. Mean recoveries were in the range 86–108%. The precision is expressed as the relative standard deviation, and in all but one of the cases was 20% or below. The accuracy of the method allows residue analyses to be performed on biological matrices at ng/g levels. The determined limit of quantification for each analyte was 8 ng/g fish. For all spiking levels ≥8 ng/g, relative standard deviations were ≤ 20%.     

A validated LC–MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study

A highly sensitive LC–MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid–liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25–500 ng/ml for SV (r² ≥ 0.9923) and 0.24–481.23 ng/ml for SVA (r² ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.     

A validated LC–MS/MS method for the quantification of capivasertib in dog plasma: Application to its pharmacokinetics study

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated using acetonitrile. An Acquity BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) maintained at 40°C was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. Multiple reaction monitoring transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. Excellent linearity was achieved in the concentration range of 1–1,000 ng/ml with a correlation coefficient of >0.9981. The lower limit of quantification was 1 ng/ml. The extraction recovery of capivasertib from dog plasma was >85.81% and no significant matrix effect was found. The intra- and inter-day precision was <9.58% and the accuracy ranged from −10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.     

A fast method for monitoring of organic explosives in soil: a gas temperature gradient approach in LC–APCI/MS/MS

A novel, fast liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC–APCI–MS/MS) screening method was developed to determine the trace amounts of TNT (trinitrotoluene), RDX (1,3,5-trinitroperhydro-1,3,5-triazine), HMX (cyclotetramethylene-tetranitramine), PETN (pentaerythritoltetranitrate), TETRYL (2,4,6-trinitrophenyl-N-methylnitramine), picric acid (2,4,6 trinitrophenol), 2,6-DNT (2,6-dinitrotoluene), and TMETN (trimethylolethane-trinitrate) which contaminate the soil after explosion. A gradient of 2.00 mM ammonium nitrate aqueous solution-methanol mobile system, C18 column, and atmospheric pressure chemical ionization (APCI) (?) ionization mode was used after a single-step solid–liquid extraction procedure from soil matrix. Phenytoin was used as the internal standard. As an extraordinary application, gas temperature gradient in an APCI ionization was used. Analytes were selectively eluted from the system within 10 min. Average recovery obtained from the soil was between 93.01 and 104.20% at 250.0, 500.0, and 1000.0 ngg^?1 concentration levels. Limit of detection (LOD) and limit of quantification (LOQ) values obtained from the analysis of the soil samples including explosive mix were between 8.9–161.2 and 13.2–241.5 ngg^?1, respectively.     

A fast and effective routine method based on HS-SPME–GC–MS/MS for the analysis of organotin compounds in biota samples

This work validated an automated, fast, and low solvent- consuming methodology suited for routine analysis of tributyltin (TBT) and degradation products (dibutyltin, DBT; monobutyltin, MBT) in biota samples. The method was based on the headspace solid-phase microextraction methodology (HS-SPME), coupled with gas chromatographic separation and tandem mass-spectrometry (GC–MS/MS). The effectiveness of the matrix-matched signal ratio external calibration was tested for quantification purposes. The exclusion of matrix influences in the calibration curves proved the suitability of this versatile quantification method. The method detection limits obtained were of 3 ng Sn g⁻¹ dw for all the analytes. The analysis of references materials showed satisfying accuracy under optimum calibration conditions (% recovery between 87–111%; |Z-scores|<2). The repeatability RSD% and intra-laboratory reproducibility RSD% were lower than 9.6% and 12.6%, respectively. The work proved the remarkable analytical performances of the method and its high potential for routine application in monitoring organotin compounds (OTC).     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.     

A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

Simultaneous quantification of ten cytotoxic drugs by a validated LC–ESI–MS/MS method

A liquid chromatography separation with electrospray ionisation and tandem mass spectrometry detection method was developed for the simultaneous quantification of ten commonly handled cytotoxic drugs in a hospital pharmacy. These cytotoxic drugs are cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. The chromatographic separation was carried out by RPLC in less than 21 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. MS/MS was performed on a triple quadrupole in selected reaction monitoring mode. The analytical method was validated to determine the limit of quantification (LOQ) and quantitative performance: lowest LOQs were between 0.25 and 2 ng mL(-1) for the ten investigated cytotoxic drugs; trueness values (i.e. recovery) were between 85% and 110%, and relative standard deviations for both repeatability and intermediate precision were always inferior to 15%. The multi-compound method was successfully applied for the quality control of pharmaceutical formulations and for analyses of spiked samples on potentially contaminated surfaces.     

Quantitative LC–MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC–MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and ?20 °C as well as after three freeze–thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.     

A facile and sensitive HPLC–MS/MS method for quantification of 3,3′-diindolylmethane in plasma samples

Indole-3-carbinol is the subject of ongoing biomedical research owing to its potential antiatherogenic, anticarcinogenic and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3′-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/ml concentration range. Consequently, the development of highly sensitive analytical methods to determine DIM in various biological samples is an urgent issue. In this study, an HPLC–MS/MS method was established for the quantification of DIM in human plasma. The developed method was validated according to the European Medicines Agency guidelines. Sensitivity, selectivity, accuracy and precision were good, allowing DIM quantification in the concentration range of 5–500 ng/ml. The limit of detection and the lower limit of quantification were 1 and 5 ng/ml, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were extracted from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C₁₈ column (50 × 2.0 mm, 4 μm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid; 85:15, v/v) was delivered at a flow rate of 0.20 ml/min. DIM and the IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to healthy female Russian volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).     

A novel LC–MS/MS method for the determination of ziritaxestat in rat plasma and its pharmacokinetic study

Ziritaxestat is a first-in-class autotoxin inhibitor. The purpose of this study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometric (LC–MS/MS) method for the determination of ziritaxestat in rat plasma. The plasma sample was deproteinated using acetonitrile and then separated on an Acquity BEH C₁₈ column with water containing 0.1% formic acid and acetonitrile as mobile phase, which was delivered at 0.4 ml/min. Ziritaxestat and the internal standard (crizotinib) were quantitatively monitored with precursor-to-product transitions of m/z 589.3 > 262.2 and m/z 450.1 > 260.2, respectively. The total running time was 2.5 min. The method showed excellent linearity over the concentration range 0.5–2000 ng/ml, with correlation coefficient >0.9987. The extraction recovery was >82.09% and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <11.20% and accuracies were in the range of −8.50–7.45%. Ziritaxestat was demonstrated to be stable in rat plasma under the tested conditions. The validated LC–MS/MS method was successfully applied to study the pharmacokinetic profiles of ziritaxestat in rat plasma after intravenous and oral administration. Pharmacokinetic results demonstrated that ziritaxestat displayed a short half-life (~3 h) and low bioavailability (20.52%).     

Development of an LC–MS/MS method for quantifying two main metabolites of abivertinib in human plasma

Abivertinib represents a highly selective irreversible epidermal growth factor receptor tyrosine kinase inhibitor. Two major metabolites of abivertinib, M7 and MII-6, were detected in human plasma, which are recommended to be monitored for safety reasons in clinical trial. A high-throughput quantification method utilizing liquid chromatography–tandem mass spectrometry was designed and verified to quantify abivertinib's primary metabolites in human plasma. Solid-phase extraction was used to process the plasma, and then the analytes underwent a gradient elution separation in an Aquity UPLC BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) with mobile phase A (10 mm ammonium acetate containing 0.1% formic acid) and mobile phase B (methanol–acetonitrile, 2:8, v/v, with 0.1% formic acid). Ion transitions of M7 (m/z 490.2 → 405.1) and MII-6 (m/z 476.2 → 391.1) were monitored under multiple reaction monitoring mode and electrospray ionization in positive ion mode. This simultaneous determination method was found to have acceptable precision, accuracy and linearity in the 0.5–500 ng/mL range for M7 and the 0.5–500 ng/mL range for MII-6, accompanied by a mild matrix effect but high recovery. Further stability assessments indicated that both analytes remained stable throughout the entire experimental process from harvesting whole blood to plasma extraction and analysis.     

A robust,accurate, sensitive LC–MS/MS method to measure indoxyl sulfate,validated for plasma and kidney cells

Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases (CKDs). Currently available diagnostic methods do not allow for early disease detection and are neither efficient. Indoxyl sulfate (IS) is an endogenous metabolite and protein-bound uremic toxin that is eliminated via renal secretion, but accumulates in plasma during tubular dysfunction. Therefore, it may be suitable as a tubular function marker. To evaluate this, a fast bioanalytical method was developed and validated for IS in various species and a kidney cell line using LC–MS/MS. An isotope-labeled IS potassium salt as an internal standard and acetonitrile (ACN) as a protein precipitant were used for sample pretreatment. The analyte was separated on a Polaris 3 C18-A column by gradient elution using 0.1% formic acid in water and ACN, and detected by negative electrospray ionization in selected reaction monitoring mode. The within-day (≤ 4.0%) and between-day (≤ 4.3%) precisions and accuracies (97.7 to 107.3%) were within the acceptable range. The analyte showed sufficient stability at all conditions investigated. Finally, applying this assay, significantly higher plasma and lower urine concentrations of IS were observed in mice with diabetic nephropathy with tubular damage, which encourages validation toward its use as a biomarker.     

Packed in-tube SPME–LC–MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC–MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2⁴⁻¹ and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME–LC–MS/MS method took another 12 min, with a sample throughput of 3 samples h⁻¹. The developed method presented adequate precision, accuracy and linearity; R² values ranged from 0.990 to 0.997, in the range of 10–1000 ng ml⁻¹. The lower limits of quantification varied from 10 to 25 ng ml⁻¹. Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.     

Rapid and cost-effective LC–MS/MS method for determination of hydroxycitric acid in plasma: Application in the determination of pharmacokinetics in commercial Garcinia preparations

Garcinia cambogia is one of the most commonly used anti-obesity dietary supplements, and hydroxycitric acid (HCA) is a major constituent in the commercial preparations of Garcinia. High doses of HCA are often consumed without much awareness of its pharmacokinetic and toxicokinetic parameters, and therefore, a complete understanding of its effects is lacking. The first step in understanding these parameters is the availability of a reliable bioanalytical method. Here, we present the first report on a UPLC–MS/MS method for analysis of HCA in rat plasma after a simplified and cost-effective protein precipitation. Chromatographic separation of the analytes in the supernatant was achieved using hydrophilic interaction liquid chromatography, where mass parameters were optimized and a rapid 5-min quantitative assay was developed. The method was highly sensitive, accurate, precise and linear in the concentration range of 10.5–5000 ng/mL (validated according to the United States Food and Drug Administration guidelines). Further, the method was successfully used to describe the pharmacokinetic profile of HCA in rat plasma after the administration of pure HCA and commercial Garcinia preparations.     

Protein precipitation method for determination of clobazam and N-desmethylclobazam in human plasma by LC–MS/MS

A protein precipitation method for the determination of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS) was established. CLB and N-CLB were extracted from human plasma samples by protein precipitation with methanol. Analyte separation was done using a Phenomenex Kinetex™ Biphenyl (50 × 2.1 mm, 1.7 μm) column using isocratic elution with a mobile phase of 5 mm ammonium formate with 0.01% ammonium hydroxide (40%) and methanol (60%) at a flow rate of 0.4 mL/min and an injection volume of 10 μL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 301.1 → 259.0, 306.0 → 263.9 for CLB and CLB-D5 and 287.0 → 245.0, 292.0 → 250.0 for N-CLB and N-CLB-D5 in positive electrospray ionization mode, respectively. The method was validated over a concentration range of 2.0–750 ng/mL for CLB and 0.7–200 ng/mL for N-CLB on SCIEX Triple Quad 4500 MS System. Total run time was 5 min. This method has been designed for bioequivalence study for formulations containing 20 mg of CLB.     

A surface wipe sampling and LC–MS/MS method for the simultaneous detection of six antineoplastic drugs commonly handled by healthcare workers

An effective wipe sampling and LC–MS/MS method was developed to simultaneously analyze six commonly administered antineoplastic drugs in stainless steel surface. The analyzed drugs were methotrexate, paclitaxel, cyclophosphamide, 5-fluorouracil, vincristine, and oxaliplatin, a frequently prepared antineoplastic drug that has not been included among any of the published simultaneous detection methods. The established method was used to evaluate the recoveries of antineoplastic drugs on brand new and worn stainless steel surfaces by wiping the plates with a Whatman filter paper wetted with 0.5 mL of water/methanol (20:80) with 0.1 % formic acid followed by LC–MS/MS before desorbing the filter with a water/methanol (50:50) solution. A significant decrease in the recovery of all evaluated drugs was found when worn plates were used. Additionally, the inter-personnel variability on drug recoveries during wiping procedures was evaluated. Significantly higher recoveries were achieved by the personnel with more training and experience versus personnel without prior experience. Finally, a laboratory stability test was developed to assess the degradation of the antineoplastic drugs during replicated shipping conditions. With the exception of vincristine sulfate which exhibited a significant (p?<?0.05) degradation after 48 h, all evaluated drugs were stable during the first 24–48 h. However, after 144 h, an increase in the degradation of all evaluated drugs was observed, with oxaliplatin and 5-fluorouracil exhibiting the most degradation.