Accurate determination of an immunosuppressant in stented swine tissues with LC–MS/MS

During stent development, accurate monitoring of the drug concentration in animal tissues can provide critical information on how the drug is released into the circulation and the surrounding tissues. To establish the relationship between the drug concentration and the distance from the stent to the target tissue, a comprehensive strategy was developed for sample collection, sample homogenization and sample storage as well as sample analysis. This strategy was developed with the analytical chemists and animal surgical specialists working together as a team. The optimized sampling process was designed to yield a representative sample, appropriately located and of an appropriate size. The sampling process was also designed to eliminate the potential for carryover and cross-contamination. During sample processing, the analyte solution was spiked into blank tissues using a sharp needle and a gas-tight syringe to prepare tissue quality control samples. These tissue quality controls were then used to evaluate the stability of the drug in solid tissue and homogenate, the homogenization carryover, the cross-contamination and the recovery of the drug during method validation and to monitor the overall process of drug analysis of the swine tissues. This thorough strategy has been applied to the accurate determination of zotarolimus in swine tissues for regulated toxicology studies. The entire process was controlled, including precise tissue sampling, compound-based tissue homogenization, method validation, and the application of the method to regulated toxicokinetics studies. The results demonstrate that analytical chemistry concepts can be successfully integrated into toxicokinetics studies in order to collect precise samples and obtain meaningful results. The strategy can be applied to similar toxicokinetics studies of locally administrated drugs in tissues.     

Adduct supported analysis of γ-hydroxybutyrate in human serum with LC-MS/MS

To avoid the detection of small fragmentation products of γ-hydroxybutyrate (GHB), a liquid chromatography–tandem mass spectrometry GHB quantification method in human serum supported by adduct formation was developed and validated. The continuous infusion of GHB/GHB-D6 made the identification of two adducts possible and GHB/GHB-D6 sodium acetate adduct fragmentation was used as target mass transition. A Luna 5 μm C18 (2) 100 A, 150 mm?×?2 mm analytical column and elution with a programmed flow of the mobile phase consisting of 10 % A (H₂O/methanol = 95/5, v/v) and 90 % B (H₂O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1 % acetic acid (pH?=?3.2), were used. Protein precipitation with 1 mL of the mobile phase B was used as the sample preparation. The calculated limit of detection/quantification was 1 μg/mL. The presented study shows that the fragmentation of GHB sodium acetate adducts is an effective way of quantification of this small molecule and is an interesting alternative to other methods based on the detection of ions smaller than 85 Da. This fact together with the short analysis time of 3 min and the fast sample preparation make this method very attractive for forensic/clinical application.     

Analysis and validation of perfluorinated compounds in water,sediment and fish with LC-ESI–MS/MS

The analysis of perfluorinated compounds (PFCs) in environmental matrices is challenging, as the concentrations are generally low, but the risk of contamination is high. Sample preparation is a critical step and it is necessary to minimise the possibility of contamination. In this study, we successfully applied and validated a modified ion pair extraction method to quantify PFCs in sediment and fish samples. A large volume injection method was validated and used to quantify PFCs in different water matrices. Isotope internal standard of every analyte was applied to correct matrix effects. The recoveries of the analytes were 92–106% for water matrix, 93–119% for fish matrix and 86–103% for soil matrix whereas the achieved limit of quantitation values were 1.3–14.9 ng/L for water, 0.19–0.28 μg/kg for fish and 0.14–0.41 for soil samples. Thirty-one surface water, 8 stormwater and 41 sediment samples collected all over Estonia were analysed and 4 (out of 8 analysed) PFCs were found in quantitative amount. The most frequently detected analyte perfluorobutanoic acid (PFBA) was found in 26% of the water samples with a maximum concentration of 137 ng/L.     

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%.     

Quantitative Analysis of Busulfan in Human Plasma by LC-MS–MS

High-performance liquid chromatography with tandem mass spectrometry has been used for rapid, specific, and sensitive analysis of busulfan in human plasma. Busulfan-d₈ was used as internal standard. Analysis was performed on a C₁₈ column (50 mm × 2.1 mm, 3.5-µm particles) with water–methanol 80:20 (v/v) as mobile phase at a flow-rate of 0.30 mL min^?1. Detection was by tandem triple–quadrupole mass spectrometry with turbo ion-spray ionization. Linear calibration plots were obtained over the concentration range 1.096–1,096 ng mL^?1. The assay is ideally suited to monitoring of busulfan and determination of its pharmacokinetic data.     

Anidulafungin—challenges in development and validation of an LC-MS/MS bioanalytical method validated for regulated clinical studies

Anidulafungin is a semi-synthetic echinocandin with antifungal activity, usually administered as an intravenous infusion. In order to determine the pharmacokinetics (PK) of anidulafungin in pediatric patients, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) bioanalytical method (M1) was developed and validated for quantification of anidulafungin in plasma. During analysis of incurred samples (samples collected from patients enrolled in a clinical study) an isobaric chromatographic interference was observed. The source of interference was identified as an anidulafungin open-ring form (D1) and its impact on the quantification of anidulafungin was investigated. It was found that accurately quantifying anidulafungin in incurred samples required chromatographic separation of the open-ring form from anidulafungin. The method was redeveloped to achieve the appropriate baseline separation and to avoid experimental conditions that favored opening the anidulafungin ring. The extraction of anidulafungin from plasma by protein precipitation remained unchanged, but the changes in chromatography warranted validation of a new method, M2, 2?years after M1 was validated. Incurred samples from three studies that were previously analyzed by M1 and were within confirmed long-term frozen stability were then reanalyzed by M2. Although the incurred sample reproducibility tests on those samples passed for each of the two methods, comparison of concentrations from the same samples obtained by M1 and M2 revealed that an overestimation of anidulafungin following the M1 method exceeded acceptance criteria. The new HPLC-MS/MS method (M2) is applicable for quantification of anidulafungin within a nominal range 50-20,000?ng/mL and requires a 50?μL human plasma aliquot. A linear, 1/concentration squared weighted, least-squares regression algorithm was used to generate the calibration curve and its parameters were used to quantitate the incurred samples. The inter-assay accuracy in heparin human plasma validation ranged from -4.33 to 0.0386?% and precision was ≤7.32?%. The method M2 was validated for use in regulated bioanalysis and is presently used to quantitate anidulafungin in plasma samples from clinical studies.     

Quantification of 33 antidepressants by LC-MS/MS—comparative validation in whole blood,plasma, and serum

In the present study, a liquid chromatography–mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid–liquid extraction was developed for fast target screening and quantification of 33 antidepressants in whole blood, plasma, and serum. The method was validated with respect to selectivity, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits. In addition, cross-calibration between the three biosamples was done to assess the impact of the different matrices on the calibration. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether–ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The approach was sensitive and selective for 33 analytes in whole blood and 31 analytes in plasma and serum and accurate and precise for 30 of the 33 tested drugs in whole blood, 31 in plasma, and 28 in serum. Cross-calibration was successful only for 13 analytes in whole blood and 16 analytes in serum calculated over a calibration curve made in plasma, 12 analytes in whole blood and 15 analytes in plasma calculated over a calibration curve made in serum, and 10 analytes in plasma and 15 analytes in serum calculated over a calibration curve made in whole blood.     

Rapid and Sensitive LC-MS/MS Method for Quantification of Fexofenadine in Human Plasma——Application to a Bioequivalence Study in Chinese Volunteers

A rapid and sensitive liquid chromatography-tandem mass spectrometry method（LC-MS/MS）was developed and validated for the quantification of fexofenadine in human plasma,to conduct comparative bioavailability studies.Human plasma was extracted with a mixture of dichloromethane-diethyl ether（volume ratio 2∶3）in a basic environment and the extract was separated on a C18 column with a mobile phase consisting of acetonitrile-methanol-10 mmol/L ammonium acetate（volume ratio 45∶45∶10）.The analytes were detected via electrospray ionization（ESI）tandem mass spectrometry in the multiple-reaction-monitoring（MRM）mode.The linearity was within a range of 1-1000 ng/mL.The intra-and inter-day precision were〈4.1% and〈4.8%,respectively,and the accuracy was in the range of 95.0%-105%.The method was applied to the quantification of fexofenadine human plasma from 20 healthy male Chinese volunteers,according to a single dose,randomized,two-way crossover design with a two-week washout period.The mean values of major pharmacokinetic parameters of ρmax,AUC0-48,AUC0-∞,tmax,and t1/2 were determined from the plasma concentration.The analysis of variance（ANOVA）did not show any significant difference between the two products of fexofenadine and 90% confidence intervals fell within the acceptable range for bioequivalence.     

Evaluation of commercial immunoassays for the detection of estrogens in water by comparison with high-performance liquid chromatography tandem mass spectrometry HPLC–MS/MS (QqQ)

In this work four different commercially available enzyme-linked immunosorbent assays (ELISA) (from Japan EnviroChemicals, Ltd., Tokyo, Japan) were evaluated in terms of performance for the rapid screening of estrogens in different water matrices, including natural and spiked samples from urban wastewater, river water and ground water. All four test kits are based on monoclonal antibodies. The compounds detected by these immunoassays are (1) 17-β-estradiol, (2) estrone, (3) 17-α-ethynyl estradiol and (4) estrogens in general, with high recognition properties for 17-β-estradiol, estrone and estriol. Standards were prepared in water containing 10% (v/v) methanol. The IC ₅₀ (corresponding to the 50% of the effective concentration) values, the dynamic ranges, and the limits of detection of the ELISA kits were 0.060–0.304 μg/L, 0.05–5 μg/L and 0.05 μg/L, respectively. All samples were extracted by solid-phase extraction (SPE) beforehand, and the evaluation was carried out by comparing the results obtained by ELISA with those obtained by HPLC–MS/MS using a triple quadrupole (QqQ) instrument. In addition, two different solid-phase extraction procedures were carried out and compared. Except for moderate overestimation in the results observed with the ELISA kits in the analysis of complex wastewater samples, the results obtained using all of the tested techniques were generally in very good agreement.      

MS／MS Study of Deoxydinucleotides Bound with Alkali—metal Ions Using ESI—MS

Positive ion ESI-MS has been used to examine the fragmentation pathways of the complex ions of deoxydinucleotides with H^ ,Na^ ,K^ by LCQ instrument.It had been found that the dissociation Varied markedly due to the differences of the base sequence.The alkali-metal ion binding site and the charcaterization of dissociation were directed by the size of metal ion,the sequesce of base and the steric hindrance.     

Identification of in vitro and in vivo metabolites of 12β-hydroxylveratroylzygadenine associated with neurotoxicity by using HPLC–MS/MS

Metabolism study was carried out on 12b-hydroxylveratroylzygadenine(VOG) that is a cevine-type alkaloid existing in Veratrum nigrum L. and a neurotoxic component. In order to better understand the potential mechanism of neurotoxicity of VOG, this study measured VOG-induced DNA damage in the cerebellum and cerebral cortex of mice after 7 days repetitive oral dose by using single-cell gel electrophoresis(Comet assay). High performance liquid chromatography-tandem mass spectrometry(LC–MS/MS) was developed and applied to separate and identify in vitro and in vivo metabolites of VOG for investing the possible relationship of metabolism and neurotoxicity. In vitro experiment was carried out using rat liver microsomes, while the in vivo study was conducted on rats. The obtained results indicated that VOG might cause DNA damage in cerebellum and cerebral cortex of mice in a dosedependent manner. Hydrolysis of ester bond and O-demethylation were proposed to be the main in vivo metabolic pathways of VOG, while the major in vitro metabolic pathways were proposed as methyl oxidation to aldehyde, dehydrogenation, hydrolysis of ester bond, hydrolysis of ester bond together with acetylation, and methoxylation. O-Demethylation reaction was likely to be associated with reactive oxygen species production, leading to the DNA damage.     

A rapid and sensitive UPLC–MS/MS method for quantitative determination of arformoterol in rat plasma,lung and trachea tissues

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and fully validated for determination of arformoterol in rat plasma, lung and trachea tissues.     

Determination of nitrophenolate sodium in aquatic products by HPLC–MS/MS with atmospheric pressure chemical ionization

The paper describes an efficient method for the determination of nitrophenolate sodium in aquatic products by HPLC combined with atmospheric pressure chemical ionization with tandem mass spectrometry (LC–APCI-MS/MS). Analytes were extracted from aquatic products by acetonitrile, the extracts were degreased by alumina and concentrated, the concentrated solution was further purified by Oasis HLB cartridge. Finally, the analytes were separated and detected by LC–APCI-MS/MS in negative ion mode. Excellent linearity with correlation coefficients of more than 0.995 was observed in the concentration range of 2–200 μg/L for p-nitrophenol sodium and 2-methoxy-5-nitrophenolate sodium, and 5–200 μg/L for ο-nitrophenol sodium. Recovery rates of nitrophenolate sodium between 86.1–94.3% were achieved. Limit of quantitation of p-nitrophenol sodium and 2-methoxy-5-nitrophenolate sodium was 2 μg/kg and ο-nitrophenol sodium was 5 μg/kg, with relative standard deviations <10%. This method was employed in the practical analysis of spiked and naturally contaminated aquatic products.     

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl—quantification by ID LC-MS/MS

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a—amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a—a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.     

Profiling of hydroxyurea-treated β-thalassemia/ serum proteome through nano-LC–ESI–MS/ MS in combination with microsol-isoelectric focusing

Advancements in proteomic tools offer a comprehensive solution to studying the complexity of diseases at molecular level. This study focusses on the clinical proteomic profiling of pre- and post-hydroxyurea (HU)-treated β-thalassemia patients in parallel with healthy individuals to better understand the role of HU in the treatment of β-thalassemia. The strategy encompasses sequential high-resolution protein fractionation using MicroSol-isoelectric focusing (ZOOM- IEF) followed by one-dimensional SDS-PAGE before nano-RP-LC–MS/ MS analysis of tryptic peptides. Protein identification was performed through Mascot search using NCBInr and SwissProt databases. Several different proteins were observed in pool serum samples of each of the three study groups. Approximately, 1250 proteins exclusive to each group were identified, and after removing the redundant and low sequence coverage proteins, the number was reduced to 576 (201 in healthy, 187 in HU-untreated and 188 in HU-treated group). Uniquely identified proteins in the HU-treated group regulate the focal adhesion, ECM-receptor interaction, PI3K-Akt signaling, Rap1 signaling, cAMP signaling, platelet activation, and Ca²⁺ signaling pathways in the HU-treated group. The proteomic profile presented here will add to the current state of understanding of molecular mechanisms involved in hydroxyurea treatment of β-thalassemia.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of steroid hormones in blood by GC–MS/MS

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08–0.16 ng/mL for estrogens, 0.20–0.36 ng/mL for androgens and 0.36–0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50–112% in the range 0.10 to 2.00 ng/mL.     

Quantification of rosiglitazone in rat plasma and tissues via LC–MS/MS: Method development,validation, and application in pharmacokinetic and tissue distribution studies

A bioanalytical method for the quantification of rosiglitazone in rat plasma and tissues (adipose tissue, heart, brain, bone, and kidney) using LC–MS/MS was developed and validated. Chromatographic separation was achieved on a Gemini C₁₈ column (50 × 4.6 mm, 3 μm) using a mobile phase consisting of 10 mM ammonium formate (pH 4.0) and acetonitrile (10:90, v/v) at a flow rate of 0.8 mL/min and injection volume of 10 μL (internal standard: pioglitazone). LC–MS detection was performed with multiple reaction monitoring mode using target ions at m/z → 358.0 and m/z → 357.67 for rosiglitazone and pioglitazone (internal standard), respectively. The calibration curve showed a good correlation coefficient (r²) over the concentration range of 1–10,000 ng/mL. The mean percentage recoveries of rosiglitazone were found to be over the range of 92.54–96.64%, with detection and lower quantification limit of 0.6 and 1.0 ng/mL, respectively. The developed method was validated per U.S. Food and Drug Administration guidelines and successfully utilized to measure rosiglitazone in plasma and tissue samples. Further, the developed method can be utilized for validating specific organ-targeting delivery systems of rosiglitazone in addition to conventional dosage forms.     

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC–MS/MS

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6 min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95–99.97 (norvancomycin), 725.90–100.04 (vancomycin), 384.16–67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5–50 μg/ml for serum and 0.5–100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.