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A rapid, sensitive, and reliable ultra-performance liquid chromatography ( UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon ( Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6-aminoquinolyl-carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C18 column with Acetonitrile-acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5-1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples. [ABSTRACT FROM AUTHOR]

Abstract: N-Phenylpropenoyl-l-amino acids (NPA) are among the key contributors to the astringent taste of cocoa. Two fast and easy to use methods (CE and UPLC®, both with PDA detection) for routine determination of the main NPA were developed. Crude extracts of defatted seeds were analysed by means of capillary electrophoresis leading to separation in less than 30min. Separation by means of UPLC® was much faster (<4min), however, a preceding SPE clean-up abolishes this benefit in time saving. Thus, the CE- and UPLC®-methods are comparable concerning time consumption and provide similar results. Analysis of 18 samples of raw and roasted beans from the global cocoa market originated from 12 countries and 4 continents showed a great variability of NPA content (0.7–3.6mg/g) and qualitative composition of different NPA. Anyway, all samples from cocoa beans showed a comparable NPA pattern. N-[3′,4′-dihydroxy-(E)-cinnamoyl]-l-aspartic acid was the most abundant metabolite, followed by N-[4′-hydroxy-(E)-cinnamoyl]-l-aspartic acid and N-[3′,4′-dihydroxy-(E)-cinnamoyl]-3-hydroxy-l-tyrosine (clovamide). The analysis of other plant organs (flowers, leaves, fruits) revealed an entirely different situation. NPA were detected in all parts of the fruit, with husk and pulp being clearly dominated by clovamide. In flowers and leaves no NPA were detected; 2-O-caffeoyltartaric acid was shown to be the major caffeic acid metabolite in leaves. [Copyright &y& Elsevier]

Abstract: The development and characterisation of a method based on reverse-phase ultra-performance liquid chromatography (UPLC) coupled to a quadrupole-time of flight mass spectrometer (Q-TOF-MS) with negative electrospray ionisation (ESI) to determine perfluorinated compounds (PFCs) in packaging is presented in this paper. Analytes were quantitatively recovered from packaging with methanol in only one PLE cycle of 6min at 100°C. The UPLC allowed the successful separation of the studied PFCs in less than 4min. The whole method presented good precision, with RSDs below 8%, LODs from 0.6 to 16ngg−1; and excellent recovery values, around 100% in all cases, were achieved. The PLE–UPLC–MS method was applied to the analysis of popcorn packaging for microwave cooking. Besides the most commonly studied PFCs: PFOA and PFOS, the presence of other perfluorocarboxylic acids (PFCAs) in popcorn packaging is evidenced in this work. [Copyright &y& Elsevier]

An Ultra Performance Liquid Chromatographic (UPLC) method for the quantification of some keto analogues of essential amino acid calcium salt has been developed. The Acquity BEH C8 column with 100 mm × 2.1 mm i.d. and 1.7 µ particle size was used to achieve the separation. The mobile phase consists of acetonitrile and 5 mM potassium di-hydrogen orthophosphate with pH = 3.0 were used with gradient elution. The flow rate was 0.4 mL/min and UV detection was performed at 215 nm. The total analysis time of all four analytes is just 8.0 min. A System Suitability Test (SST) was developed to govern the quality of the separation. The developed method has been validated further with respect to linearity, accuracy, precision, selectivity, LOD, LOQ, and robustness. Different batches of samples were examined using this method. Furthermore, the method proved to be successful when applied to analyze a marketed formulation. [ABSTRACT FROM AUTHOR]

Abstract: We developed a stable isotope dilution ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-d 4 and cotinine-d 4 as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5–35,000μg/L (r >0.999). Limits of Detection were 0.7μg/L for nicotine and 0.4μg/L for cotinine, and Lower Limits of Quantification were 1.7μg/L for nicotine and 1.1μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were <4% and <2%, respectively, the interassay CVs were <6% for nicotine and <4% for cotinine. The inaccuracy was <6% for both substances. The mean recovery was 103.2% (range 96.8–105.1%) for nicotine and 97.4% (range 94.3–99.2%) for cotinine. A method comparison showed that the values of nicotine metabolites in human urine samples (n =98) measured by a commercially available chemiluminescent immunoassay tested on analyzer IMMULITE 2000 were much higher than the cotinine concentration in the same urine samples measured by our UPLC–MS/MS assay. The Passing–Bablok regression line was: immunoassay=4.62 (UPLC–MS/MS)+3.64 [μg/L]; r =0.75. This robust, sensitive and interference-free UPLC–MS/MS assay permits rapid and accurate determination of nicotine and cotinine in human urine. [Copyright &y& Elsevier]

Abstract: Procainamide, a type I antiarrhythmic agent, is used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25–30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice as well as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation. [Copyright &y& Elsevier]

Surfactants and their metabolites can be found in aquatic environments at relatively high concentrations compared with other micropollutants due in part to the exceptionally large volumes produced every year. We have focused our attention here on the most widely used nonionic surfactants, alcohol ethoxylates (AEOs), and on nonylphenol ethoxylate (NPEO) degradation products (short-chain nonylphenol ethoxylates, NP1-3EO, nonylphenol, NP, and nonylphenol ethoxycarboxylates, NP1-2EC), which are endocrine-disrupting compounds. Our main objective in this work was to develop a methodology aimed at the extraction, isolation, and improved analysis of these analytes in environmental samples at trace levels. Extraction recoveries of target compounds were determined for sediment samples after ultrasonic extraction and purification using HLB or C18 solid-phase extraction minicolumns. Recovery percentages were usually between 61 and 102% but were lower for longer AEO ethoxymers. Identification and quantification of target compounds was carried out using a novel ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS-MS) approach, a combination that provides higher sensitivity and faster analysis than prior methods using conventional high-performance liquid chromatography-mass spectrometry. Limits of detection were usually below 0.5 ng/g, being higher for monoethoxylate species (>5 ng/g) because of poor ionization. The method was used for analyzing surface sediment samples collected at Jamaica Bay (NY) in 2008. The highest values (28,500 ng/g for NP, 4,200 ng/g for NP1-3EO, 22,400 ng/g for NP1-2EC, and 1,500 ng/g for AEOs) were found in a sampling station from a restricted water circulation area that is heavily impacted by wastewater discharges. [ABSTRACT FROM AUTHOR]

Skeletal remains of a domestic pig were assessed for relative distribution of amitriptyline, citalopram, and metabolites. Following acute exposure and outdoor decomposition for 2 years, drugs and metabolites were analyzed in 13 different bones. Bones were pulverized following a simple wash procedure, and drugs were extracted by passive incubation in methanol, followed by solid-phase extraction. Samples were analyzed by ultra-high performance liquid chromatography (UHPLC) and confirmed with gas chromatography-mass spectrometry. The Kruskall-Wallis test showed that bone type was a main effect with respect to drug level for all analytes, with levels varying from 33- to 166-fold. Ratios of levels of drug to that of the corresponding metabolite were less variable, varying roughly one- to eightfold. This suggests limitations in the interpretive value of drug measurements in bone and that relative levels of drug and metabolites should be further investigated in terms of forensic value. [ABSTRACT FROM AUTHOR]

A rapid, specific, and sensitive ultra-performance liquid chromatographic (UPLC) method for quantitative analysis of raloxifene in bulk drug and pharmaceutical formulations has been developed and validated as per the guidelines laid by ICH. The chromatographic separation of raloxifene was achieved on a Waters Acquity BEH C18, 50 × 2.1 mm, 1.7 µm column with mobile phase containing a gradient mixture of solvent A (0.1% of Formic acid in water) and solvent B (acetonitrile). The method was linear in the range of 0.1–50 µg/mL (r2 = 0.9998) and having a run time of 6 min, which allows for a large number of samples to be analyzed in less time. The analyte was monitored at 287 nm. The method was validated according to the ICH guidelines with respect to linearity, precision, accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), specificity, and robustness. Forced degradation studies were also performed for raloxifene bulk drug samples to demonstrate the stability-indicating aspect of the UPLC method. Degradation was found to occur in reductive condition only while the drug was stable to oxidation, alkaline, acidic, photolytic, and hydrolytic stress conditions. The developed method was applied for the assay of raloxifene formulations like tablets and nanoparticulate formulation. [ABSTRACT FROM PUBLISHER]

Abstract: The resolution of free dl-amino acids in human nail was carried out by combination of the R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(−)-DBD-PyNCS] derivatives and UPLC–ESI-TOF-MS. The reaction of the reagent with amino acids effectively proceeds at 55°C for 20min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers. Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H2O and CH3CN containing 0.1% formic acid (HCOOH) or 5mM CH3COONH4 and CH3CN) using a reversed-phase ACQUITY UPLC™ BEH C18 (1.7μm, 100mm×2.1mm i.d.) column and sensitively detected by TOF-MS. The detection limits (S/N=3) of the TOF-MS were 1.0–750fmol, respectively. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 6-aminohexanoic acid, versus the injected amounts of each amino acid (r 2 >0.996), and the intra-day and inter-day assay precisions were less than 8.93%. The derivatives of the free dl-amino acids in human nail were successfully identified by the proposed procedure. As we know, for the first time, these five kinds of d-amino acids, which were d-Ala, d-Val, d-Pro, d-Ile and d-Leu, were found from human nail samples. Fifteen kinds of l-amino acids were also recognized from human nails. Using these methods, the amounts of dl-amino acids in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from diabetic patients to those from healthy volunteers, there is no significant difference in the content of the l-amino acids in the nails. However, a statistically significant (P <0.01) correlation was observed between the d/l-amino acid concentration ratios (Ala, Val, Ile, Leu). Therefore, because the proposed method provides a good mass accuracy and the trace detection of the dl-amino acids in human nails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients. [Copyright &y& Elsevier]

Abstract: Hyphenation of micro-liquid and ultra-performance liquid chromatography with a hybrid (QqTOF) tandem mass spectrometry was studied from the viewpoint of their applicability for monitoring of anthocyanin dyes in various cultivars and different vintages of red wine. After appropriate optimization both techniques proved to be suitable for this task. UHPLC system provided lower LOD and LOQ values as well as higher productivity and precision of retention parameters and peak areas with respect to micro-LC method. On the other hand, micro-LC method offers significant savings of mobile phase, less contamination of ion source of mass spectrometer and makes the nebulization process in electrospray much easier. In combination with principal component analysis (PCA), the method allows to elucidate long-term relations in a complete set of certified red wine cultivars grown in South Moravia (certified in Czech Republic until year 2005). Among the studied varieties Rubinet exhibit a specific anthocyanin profile. Especially interesting is a high content of 3-coumaroylglucoside-5-glucosides of malvidin and peonidin. Those dyes can be used as markers of artificial color enhancement. Principal component analysis applied to data processed with regard to enzymatic activity markedly facilitate classification among varieties. This approach allows resolution of teinturiers, variety Blaufränkish and partial classification of other varieties. [Copyright &y& Elsevier]

A comprehensive two-dimensional system coupling ultra-performance liquid chromatography (UPLC) and ion mobility-mass spectrometry (IM-MS) has been applied for the separation and analysis of hydroxylated polybrominated diphenyl ethers (OH-PBDEs). A complex mixture containing 23 OH-PBDE congeners ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl ether (OH-octaBDE) was satisfactorily separated within 16 min of analysis time. The first-dimensional reversed-phase UPLC was performed on a sub-2 μm BEH C chromatographic column using acetonitrile-water gradient elution program with a flow rate ramp. It enabled excellent chromatographic separation for both between-class and within-class OH-PBDEs based on their differences in hydrophobicity. Following the pre-ionization resolution in the first dimension, the second-dimensional IM-MS employed a hybrid electrospray quadrupole ion mobility time-of-flight mass spectrometer and added an extra post-ionization separation for between-class OH-PBDE congeners on account of their relative mobility disparity during a very short period of 8.80 ms. The orthogonality of the developed two-dimensional system was evaluated with the correlation coefficient of 0.9665 and peak spreading angle of 14.87°. The peak capacity of the system was calculated to be approximately 2 and 15 times higher than that of the two dimensions used alone, respectively. The two-dimensional separation plane also contributed to the removal of background interference ions and the enhanced confidence in the characterization of OH-PBDEs of interest. [ABSTRACT FROM AUTHOR]

Abstract: A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities. [Copyright &y& Elsevier]

Abstract: A sensitive and reliable method was developed and validated for the analysis of ractopamine in swine tissues. The sample preparation procedure was based on hydrolysis overnight, extraction with ethyl acetate, and solid-phase extraction cleanup. The target compound was determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. The average recoveries ranged between 93.3% and 106.5%, with relative standard deviations of 7.2–12.8%. The detection and quantification limits were 0.2 and 0.5μg/kg, respectively. The depletion profile of ractopamine was studied in healthy pigs after oral administration of feed containing 20mg/kg ractopamine for 30 consecutive days. Ractopamine residues were still detected 11days post-medication in all tissues examined with the exception of muscle. The highest ractopamine level was found in lung tissue. The developed method has been successfully applied to the depletion study of ractopamine in swine tissues. [Copyright &y& Elsevier]

Abstract: A reversed-phase ultra-performance liquid chromatographic (UPLC) method is described for the simultaneous determination of aldoses and uronic acids. The separation was carried out on a UPLC™ HSS T3 column (100mm×2.1mm, 1.7μm) using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and UV detection at 250nm, and 9 PMP derivatives of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose were baseline separated within 5.5min. Furthermore, the described method was successfully applied to the quantitative analysis of component monosaccharides in polysaccharides extracted from traditional Chinese medicines (TCMs). The results demonstrated the proposed UPLC method was precise and practical for the analysis of TCM polysaccharides. Finally, principal component analysis (PCA) and linear discriminant analysis (LDA) were used to classify and differentiate 30 polysaccharide samples from five TCMs to provide the basis for guiding reasonable use of TCM polysaccharides and controlling their quality better. [Copyright &y& Elsevier]

Abstract: A method to classify extra virgin olive oils (EVOOs) according to their genetic variety using sterol profiles obtained by ultra-performance liquid chromatography (UPLC) with atmospheric pressure chemical ionization mass spectrometry (APCI-MS) detection has been developed. The optimal separation conditions were obtained using a gradient acetonitrile/water (0.01% acetic acid) at a flow rate of 0.8mLmin−1 and 10°C. Linear discriminant analysis (LDA) models were constructed with the 11 UPLC-APCI-MS sterol peaks taken from the selective ion recording mode chromatograms. Ratios of the peak areas selected by pairs were used as predictors. With the sequential application of two LDA models and using a 95% probability, the EVOO samples belonging to seven genetic varieties mainly produced at La Comunitat Valenciana, Spain, were correctly classified with a prediction capability higher than 97%. [ABSTRACT FROM AUTHOR]

Ultra-performance liquid chromatography (UPLC) in reversed-phase (RP), ion pair (IP) and hydrophilic interaction chromatography (HILIC) has been investigated for the separation of imidazolium-based ionic liquid (IL) cations. Among the three stationary phases (i.e., C18, C8 and phenyl) studied under RP conditions the phenyl phase provided much stronger retention for the IL cations. Four acids (hydrochloric, methanesulfonic, perchloric and trifluoroacetic) as mobile phase additives were compared in light of their effects on the retention of IL cations. It was shown that the retention of all IL cations decreased upon acidification of the mobile phase, possibly due to suppression of residual silanol ionization. Very fast (~3 min) and efficient RP-UPLC separation of six cations was achieved by gradient elution with acetonitrile-water mobile phase containing 2.5 mmol L perchloric acid. In IP-UPLC all solutes were well resolved in about 4 min by gradient elution with acetonitrile-water mobile phase containing 1 mmol L sodium 1-octanesulfonate as ion pairing reagent. Finally, under HILIC conditions by using isocratic elution with acetonitrile-water (85:15, v/v) mobile phase containing 5 mmol L ammonium formate (pH 3.2) the separation time was reduced to less than 2 min while maintaining excellent peak shapes and sufficient resolution. Compared to current LC systems UPLC allowed considerably faster separations with better peak shapes. [ABSTRACT FROM AUTHOR]

Abstract: Ifosfamide (IF) and cyclophosphamide (CP) are common chemotherapeutic agents. Interestingly, while the two drugs are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS), and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified by UPLC–ESI-QTOFMS for both IF and CP, five were found to be novel. Ifosfamide preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while cyclophosphamide preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, a multi-residue method based on a bag-solid phase extraction (bag-SPE) technique was evaluated for determination of 10 pharmaceuticals in surface water close to the effluent of a sewage treatment plant (STP) and along a coastal gradient from a STP effluent. The 10 compounds selected were caffeine, atenolol, metoprolol, oxazepam, carbamazepine, ketoprofen, naproxen, ibuprofen, diclofenac and gemfibrozil. All analyses were performed using ultra-performance liquid chromatography (UPLC) combined with quadrupole time-of-flight (QTOF) mass spectrometry. The detection limits (LOD) ranged from 1.0 to 13ngL−1. The method showed linear concentration ranges from 25 to 800ngL−1 with regression coefficients (R 2) better than 0.9801. The recoveries of the selected analytes ranged from 11 to 65% with relative standard deviations (RSD) of <16% and inter-day variations of less than 18%. Isotopically labeled surrogate standards were used to compensate for sampling losses and matrix effects. Four of the selected 10 pharmaceuticals (caffeine, metoprolol, oxazepam and carbamazepine) were quantified, at concentrations ranging from 4 to 210ngL−1. [Copyright &y& Elsevier]

A high-throughput ultra-performance liquid chromatography (UPLC) method with UV detection was elaborated and validated for the simultaneous quantification of flunitrazepam and its major metabolite, 7-aminoflunitrazepam, in human plasma. After a simple liquid-liquid extraction with ethyl acetate, chromatographic separation was performed on a BEH C18 chromatographic column with a binary mixture of acetonitrile/10 mM potassium monohydrogen phosphate (pH = 8.5) as the mobile phase, under gradient elution (flow rate = 0.275 mL/min). The total run time of the chromatographic analysis was 4.2 min. UV detection was performed at 314 nm for flunitrazepam and at 242 nm for 7-aminoflunitrazepam. To our knowledge, this is the first reported UPLC-PDA method for the quantification of flunitrazepam and 7-aminoflunitrazepam in human plasma. The assay was applied successfully for the analysis of the selected two analytes in real human plasma samples obtained from a healthy volunteer, after ingestion of a single dose of flunitrazepam. [ABSTRACT FROM AUTHOR]

Abstract: Several freshwater cyanobacteria species have the capability to produce toxic compounds, frequently referred to as cyanotoxins. The most prevalent of these cyanotoxins is microcystin LR. Recognizing the potential health risk, France, Italy, Poland, Australia, Canada, and Brazil have set either standards or guidelines for the amount of microcystin LR permissible in drinking water based on the World Health Organization guideline of one μg/L of microcystin LR. Recently, the United States Environmental Protection Agency has begun to evaluate the occurrence and health effects of cyanotoxins and their susceptibility to water treatment under the Safe Drinking Water Act through the Contaminant Candidate List (CCL). A recent update of the Contaminant Candidate List focuses research and data collection on the cyanotoxins microcystin LR, anatoxin-a, and cylindrospermopsin. Liquid Chromatography/Tandem-Mass Spectrometry (LC/MS/MS) is a powerful tool for the analysis of various analytes in a wide variety of matrices because of its sensitivity and selectivity. The use of smaller column media (sub 2μm particles) was investigated to both improve the speed, sensitivity and resolution, and to quantify the CCL cyanotoxins, in a single analysis, using Ultra-Performance Liquid Chromatography (UPLC®) combined with tandem mass spectrometry. Natural waters and spiked samples were analyzed to show proof-of-performance. The presented method was able to clearly resolve each of the cyanotoxins in less than eight minutes with specificity and high spike recoveries. [Copyright &y& Elsevier]

Abstract: Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG). The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05–100 (r >0.999)mgL−1 and 4–4000mgL−1 (r >0.999), respectively. Limits of Detection were 0.014mgL−1 for MPA and 1.85mgL−1 for MPAG. Lower Limits of Quantification were 0.05mgL−1 for MPA and 2.30mgL−1 for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1–129.7%) for MPA and 111.1% (range 73.0–139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS=1.14 UPLC-MS/MS—0.14 [mgL−1], r =0.96, and HPLC-MS/MS=0.77 UPLC-MS/MS+0.50 [mgL−1], r =0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids. [Copyright &y& Elsevier]

Abstract: A rapid, sensitive and specific ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–QToF-MS) method has been developed and validated for simultaneous determination of three tetracyclines (TCs) including oxytetracycline, tetracycline, and chlortetracycline in human urine. Human urine sample preparation involves pH adjustment to 4 with hydrochloric acid, pre-concentration, and cleanup by solid phase extraction (SPE) process. The chromatographic separation of all TCs was achieved in less than 5min using UPLC and ESI-QToF-MS was successfully employed for the identification and quantification. The accurate masses of the product ions were calculated using lock mass correction, and were deviated from the theoretical masses by 0.5–1.9mDa and 1.1–4.5ppm, respectively. The developed method was validated in terms of linearity, sensitivity, selectivity, decision limit, detection capability, accuracy, and precision. Limit of detection and limit of quantitation for all TCs were estimated in the range of 0.089–0.138ngmL−1 and 0.294–0.455ngmL−1, respectively. High overall recoveries of greater than 90% were achieved with linear responses over the 0.5–2ngmL−1 range for all TCs in urine samples. [Copyright &y& Elsevier]

Three different instrumental methods have been used to examine the organosulfur chemistry of intact and cut garlic and onions: X-ray fluorescence spectroscopic imaging (XFS), direct analysis in real time (DART) mass spec trometry, and ultra-performance liquid chromatography-(Ag+)-coordination ion spray mass spectrometry (UPLC-(Ag+)CIS-MS). The first technique has been used to map the location of different chemical forms of sulfur in intact and damaged onion cells, the second technique, to identify the reactive, volatile sulfur compounds formed on cutting the plants, and the third technique, to identify members of families of polysulfides found in the distilled oil of garlic. [ABSTRACT FROM AUTHOR]

Abstract: Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2μm glycan column for the separation of 2-aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7μm amide sorbent improves the peak capacity compared to a 3.0μm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2–0.5mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time. [Copyright &y& Elsevier]

Abstract: Reverse-phase ion-pair high performance liquid chromatography (RPIP-HPLC) and ultra-performance liquid chromatography (RPIP-UPLC) are increasingly popular chromatographic techniques for the separation of organic compounds. However, the fine details of the RPIP separation mechanism are still being debated. Many factors including type and concentration of the ion-pairing reagent, mobile phase pH, organic modifier, ionic strength, and stationary phase all play a role in the overall efficiency and optimization of ion-pairing separations. This study investigates the role that competition between ion-pairing reagents with different steric bulk and hydrophobicity plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides. In addition to providing insights into the mechanism by which RPIP-HPLC can resolve closely related disaccharides, the use of competition between ion-pairing agents could lead to new methods for the separation of larger heparin and HS oligosaccharides. This approach should also be applicable to the analysis of other compound classes, and could lead to a general approach for the chromatographic resolution of mixtures of charged analytes having similar structures. [Copyright &y& Elsevier]

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 “designated substances” (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-( N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC™ BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile–methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H]+ obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection $$ \left( {{\text{S/N}} = 3} \right) $$ and the limits of quantification $$ \left( {{\text{S/N}} = 10} \right) $$ in 0.1 mL of human plasma and urine for the present method were 0.30–150 pmol and 1.0–500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A simple and sensitive method for determination of the O-demethylation activity of rat, dog, minipig, and human liver micrsomes toward paeonol using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized O-demethylated metabolite of paeonol (2,4-dihydroxyacetophenone, DHA) as a standard for method validation. Validation was done with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50mm×2.1mm i.d., 1.7μm), with phase of acetonitrile–water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for DHA was linear from 0.5–100μM with r 2 =0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in paeonol O-demethylation (709.7pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6pmol/min/mg protein), HLMs (569.3pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3pmol/min/mg protein). The developed method was appropriated for rapid screening paeonol O-demethylation activity in liver microsomes from different species. [Copyright &y& Elsevier]

Abstract: A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography–mass spectrometry (GC–MS) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC–MS/MS, which was completed in 5min for each injection. For the GC–MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC–MS and UPLC–MS/MS was 10 and 5μgkg−1, respectively. This validated method was successfully applied to the determination of melamine in real samples from market. [Copyright &y& Elsevier]

Abstract: Withdrawal of the support for the REMEDi HS drug profiling system has necessitated its replacement within our laboratories with an alternative broad toxicological screening technique. To this end, a novel method, based on ultra-performance liquid chromatography (UPLC) and time-of-flight (TOF) mass spectrometry, was developed for the routine analysis of urine samples. Identification was achieved by comparison of acquired data to libraries containing more than 300 common drugs and metabolites, and was based on a combination of retention time, exact mass and fragmentation patterns. Validation data for the method is presented and comprised an evaluation of the following parameters: precision; transferability of the methodology between the six collaborating laboratories; specificity; extraction recovery and stability of processed samples; matrix effects and sensitivity. This paper presents the benefits of supplementary fragmentation data with particular regard to increasing specificity and confidence of identification and its usefulness with overdosed samples. The utility of the method was assessed by the parallel analysis of 30 authentic urine samples using the REMEDi HS and UPLC–TOF. The latter provided enhanced detection, leading to the identification of twice as many drugs. Furthermore it did not miss any compounds that were identified by REMEDi HS. The UPLC–TOF findings were further verified by a combination of data from three other conventional screening techniques, i.e., GC–MS, HPLC-DAD and UPLC–MS/MS. [Copyright &y& Elsevier]

A rapid, sensitive and specific ultra performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry method was developed and validated for the determination of clopidogrel in dog plasma. Plasma samples were deproteinized with acetonitrile and separated on a Waters BEH C18 column (1.7 μm, 50 mm × 2.1 mm id.) with isocratic elution at a flow-rate of 0.2 mL min−1 and mobile phase consisting of acetonitrile and water (containing 0.15% formic acid) (75:25, v/ v). The single run analysis was as shorter as 2 min. Electrospray ionization in positive ion mode and multiple reaction monitoring were used for the quantification of clopidogrel with monitored transitions m/z 322 → 212 for clopidogrel and m/z 324 → 217 for internal standard (gliclazide). The intra- and inter-day precisions (RSD%) were less than 6.32 and 7.03%, and accuracy (RE%) between −9.12 and 9.65% ( n = 6). The extraction recovery of clopidogrel was 96.7%. The developed method was successfully applied to the pharmacokinetic study of clopidogrel tablets in dogs following oral administration at a single dose of 75 mg. [ABSTRACT FROM AUTHOR]

Abstract: An ionic liquid-based ultrasonic-assisted extraction (ILUAE) method has been developed for the effective extraction of piperine from white pepper powder. A series of 1-alkyl-3-methylimidazolium ionic liquids differing in composition of alkyl chain and anion were evaluated for extraction efficiency. The results indicate that both the characteristics of anions and cations have remarkable effects on the extraction efficiency. In addition, the ILUAE procedure was also optimized on some ultrasonic parameters, such as ultrasonic power, extraction time and solid–liquid ratio. Compared with the conventional heat-reflux extraction (HRE) and regular UAE, the optimized approach gained the highest extraction efficiency (from 1.950% to 3.577%) within the shortest extraction time (from 2h to 30min). Furthermore, the developed method was validated by the recovery, correlation coefficient (R 2), and reproducibility (relative standard deviation (RSD), n =3). Average recoveries of piperine were between 93.5% and 98.7% at spiking levels 0.05, 0.1 and 0.2mgmL−1 with RSD lower than 5%. The developed method is linear at concentrations within the tested interval, with R 2 at 0.9994. [Copyright &y& Elsevier]

Abstract: A new strategy of target optimization was developed for resolving the separation problems of complex samples. Computer-aided target optimization for separation of complex samples by ultra-performance liquid chromatography (UPLC) was studied. Taking traditional Chinese medicine (TCM) (Rhizoma Coptidis) as an example, separation conditions of target compounds – overlapping peaks and all components – were optimized based on moved overlapping separation ranging map (OSRM) and artificially intervention. After calculating retention parameters and peak shape parameters of the target peaks, the optimization was operated in an emulational picture through the computer without further experiments. The emulational chromatogram was proved to be authentic by the estimated values, which were almost identical to experimental results. The method was very helpful for obtaining the satisfied separation conditions of target compounds rapidly and efficiently. [Copyright &y& Elsevier]

Abstract: A rapid, sensitive and reliable multi-residue method for the simultaneous determination of four 5-nitroimidazoles (NIIMs) and their three corresponding metabolites in swine kidney was developed and validated. The compounds of interest were extracted from tissues with ethyl acetate. The crude extracts were subject to liquid–liquid partition with hexane followed by solid-phase extraction using mixed-mode strong cation-exchange column. Chromatographic separation was achieved on an AcQuity BEH C18 column and was completed within 4min for each injection. Data acquisition under positive electrospray tandem mass spectrometry was performed by applying multiple reaction monitoring for both identification and quantification. Mean relative recoveries from fortified samples ranged from 83% to 111%, with coefficients of variation lower than 12%. The limits of detection and quantification for the NIIMs were in the range of 0.05–0.5 and 0.1–0.5μgkg−1, respectively. [Copyright &y& Elsevier]

A simple and rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry method for the simultaneous determination of thirteen free fatty acids (FFAs) in Pheretima has been developed and validated. Measurements for each FFA were linear over a wide range (0.05–3.95 μg mL−1) with good correlation coefficients (>0.99). The limit of detection and limit of quantification for all the fatty acids were below 26 and 78 ng mL−1, respectively. The intra- and inter-assay precision and accuracy for the thirteen FFAs fell well within the predefined limits of acceptability. Satisfactory recoveries were in the range of 96–103%. [ABSTRACT FROM AUTHOR]

• Assay for simultaneous determination of 5THP and 3OMD in dried blood spot (DBS). • 3OMD and 5THP assay by UPLC-MS/MS method is sensitive, accurate and precise. • A dynamic reference values in function of age was established. • 5THP and 3OMD accumulates in DBS in aromatic L-amino acid decarboxylase deficiency. 5-hydroxytryptophan (5HTP) and 3-O-methyldopa (3OMD) are CSF diagnostic biomarkers of the defect of aromatic L-amino acid decarboxylase (AADC), a rare inherited disorder of neurotransmitter synthesis which, if untreated, results in severely disabling neurological impairment. In the last few years, different methods to detect 3OMD in dried blood spot (DBS) were published. We developed and validated a fast and specific diagnostic tool to detect 5HTP alongside 3OMD. After extraction from DBS, 3OMD and 5HTP were separated by ultra-performance liquid chromatography (UPLC) and detected by tandem mass spectrometry (MS/MS). Instrument parameters were optimized to obtain the best sensitivity and specificity. Chromatographic separation was accomplished in 13 min. The limit of detection was 2.4 and 1.4 nmol/L of blood for 3OMD and 5HTP respectively, and response was linear over the blood range of 25–5000 nmol/L. Between-run imprecision was less than 9% for 3OMD and <13% for 5HTP. An age-specific continuous reference range was established, revealing a marked and continuous 3OMD decline with aging. The effect of age on 5HTP was less evident, showing only a slight decrease with age after the first week of life. A marked increase of both 3OMD and 5HTP was found in four patients affected by AADC deficiency (1780.6 ± 773.1 nmol/L, rv 71.0–144.9; and 94.8 ± 19.0 nmol/L, rv 15.2–42.8, respectively) while an isolated increase of 3OMD (6159.6 ± 3449.1 nmol/L, rv 73.2–192.2) was detected in three subjects affected by inherited disorders of dopamine synthesis under levodopa/carbidopa treatment (a marginal increase of 5HTP was detected in one of them). Simultaneous measurement of 5HTP and 3OMD in DBS leads to an improvement in specificity and sensitivity for the biochemical diagnosis of AADC deficiency. [ABSTRACT FROM AUTHOR]

A novel and efficient stability-indicating, reverse phase ultra-performance liquid chromatographic (UPLC®) analytical method was developed and validated for the determination of hexoprenaline in an injectable dosage form. The development of the method was performed using analytical quality by design (AQbD) principles, which are aligned with the future requirements from the regulatory agencies using AQbD principles. The method was developed by assessing the impact of ion pairing, the chromatographic column, pH and gradient elution. The development was achieved with a Waters Acquity HSS T3 (50 × 2.1 mm i.d., 1.8 µm) column at ambient temperature, using sodium dihydrogen phosphate 5 mM + octane-1-sulphonic acid sodium salt 10 mM buffer pH 3.0 (Solution A) and acetonitrile (Solution B) as mobile phases in gradient elution (t = 0 min, 5% B; t = 1 min, 5% B; t = 5 min, 50% B; t = 7 min, 5% B; t = 10 min, 5% B) at a flow rate of 0.5 mL/min and UV detection of 280 nm. The linearity was proven for hexoprenaline over a concentration range of 3.50–6.50 µg/mL (R2 = 0.9998). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability and injectable dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with enhanced knowledge, which can eventually lead to a reduced regulatory risk, high quality data and lower operational costs. [ABSTRACT FROM AUTHOR]

Abstract: A method based on ultra-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (UPLC–MS/MS) for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with Decision 2002/657/EC. Sample extraction was evaluated in methanol and in ethyl acetate, the latter which gave better results. Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation chromatography (GPC). Recoveries ranged from 40% to 79% for the SPE approach and from 80% to 109% for GPC. Quantification was performed with blank tissue samples spiked with the analytes in the range 50–500μgkg−1. 5,6-Dimethyl-2-thiouracil and 2-mercaptobenzimidazole-d 4 were used as internal standards. Decision limit (CCα) and detection capability (CCβ) ranged from 1 to 15μgkg−1 and from 6 to 25μgkg−1, respectively. The accuracy of the method was calculated as percent error, which was less than 10%. The relative standard deviation in reproducibility conditions ranged between 2% and 14%. [Copyright &y& Elsevier]

Abstract: The rapid, sensitive and simultaneous determination of six polyamines, i.e., ornithine (ORN), 1,3-diaminopropane (DAP), putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), in human hairs was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS). The primary (–NH2) and secondary (–NH) amines in the polyamine structures were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60°C for 30min in the mixture of 0.1M borax (pH 9.3) and acetonitrile (CH3CN). The resulting derivatives were perfectly separated using an ACQUITY UPLC™ BEH C18 column (1.7μm, 100mm×2.1mm i.d.) by a gradient elution with a mixture of water-acetonitrile containing 0.1% formic acid (HCOOH). The separated polyamine derivatives were sensitively detected with both FL and TOF-MS. The detection limits in FL and TOF-MS were 11–86 and 2–5fmol, respectively. However, the determination of several polyamines by FL detection was interfered with by endogenous substances in the hair. Therefore, the simultaneous determination in hair was carried out by the combination of UPLC separation and the ESI-TOF-MS detection. The structures of the polyamines were identified from the protonated-molecular ions [M+H]+ obtained from the TOF-MS measurement. A good linearity was achieved from the calibration curves, that was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 1,6-diaminohexane (DAH), against the injected amounts of each polyamine (0.05–50pmol, r 2 >0.999). The proposed method was applied to the determination in the hairs of healthy volunteers. The mean concentrations of ORN, DAP, PUT, CAD, SPD and SPM in 1mg of human hairs (n =20) were 1.46, 0.18, 1.18, 0.11, 1.97 and 0.98pmol, respectively. Because the proposed method provides a good mass accuracy and the trace detection of the polyamines in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair. [Copyright &y& Elsevier]

Abstract: The application of reversed-phase ultra-performance liquid chromatography, based on the use of sub 2μm particles, combined with time-of-flight mass spectrometry has been investigated for the production of global metabolite profiles from human urine. The stability and repeatability of the methodology, which employed gradient elution, was determined by the repeat analysis of a pooled quality control (QC) sample. As seen in previous studies conducted with conventional LC–MS an element of system conditioning was required to obtain reproducible data, as the initial injections were unrepresentative. However, once the system had equilibrated excellent repeatability in terms of retention time, signal intensity and mass accuracy was seen providing confidence that for this matrix, the within-day repeatability of UPLC–TOF-MS was sufficient to assure data quality in global metabolic profiling applications. [Copyright &y& Elsevier]

A rapid, specific, and sensitive ultra-performance liquid chromatographic method for analysis of levofloxacin in human plasma has been developed and validated. Plasma samples were spiked with the internal standard (enoxacin) and extracted with 10:1 ( v/ v) ethyl acetate–isopropanol. UPLC was performed on a 100 × 2.1 mm i.d., 1.7 µm particle, C18 column with 88:12 ( v/ v) 0.4% triethylamine buffer (pH 3)–acetonitrile as mobile phase, pumped isocratically at a pressure of 11,000 psi (758 bar) and a flow-rate of 0.3 mL min−1. Ultraviolet detection was performed at 300 nm. The retention times of levofloxacin and enoxacin were 3.4 and 2.8 min, respectively, and the run-time was 5 min. Calibration showed that response was a linear function of concentration over the range 0.05–10 µg mL−1 ( r 2 ≥ 0.99) and the method was validated over this range for both precision and accuracy. The relative standard deviation was <15% for both intra-day and inter-day assay ( n = 5). Levofloxacin and enoxacin were stable in plasma; there was no evidence of degradation during three freeze–thaw cycles, post-preparative stability at 20 °C was ≥24 h, short-term stability at room temperature was ≥6 h, and long-term stability at −70 °C was ≥30 days. The method was successfully used in a study of the bioequivalence of two levofloxacin tablet formulations in healthy volunteers. [ABSTRACT FROM AUTHOR]

Abstract: A rapid and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3′-azido-3′-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50mm×2.1mm i.d., 1.7μm), with phase of acetonitrile–water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5–500μM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4°C for at least 72h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity. [Copyright &y& Elsevier]

The occurrence of several psychoactive drugs in water resources from north-eastern Spain (NE-Spain) has been evaluated. The drugs were analyzed using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–ESI-MS/MS) after enrichment by solid-phase extraction (SPE). Most of the studied controlled drugs (8 out of 11) were found in both influent and effluent samples from several wastewater treatment plants. Cocaine and its metabolite were detected in wastewaters at concentrations ranging from 4 ng/L to 4.7 μg/L and from 9 ng/L to 7.5 μg/L respectively while concentrations of amphetamine type stimulatory drugs ranged from 2 to 688 ng/L. Removal percentages were estimated by sampling eight WWTPs (n =4). Cocaine and benzoylecgonine removal percentages were higher than 88% while those of amphetamine type stimulants varied ranging from 40% to more than 99%. Daily variability was also evaluated by performing a sequential survey, which revealed important fluctuations in the concentrations of nicotine, paraxanthine, amphetamine and ecstasy during the week. From the total concentrations found in wastewater influents estimations of the cocaine and ecstasy consumption were performed. For cocaine the results were approximately 14 doses per 1000 inhabitants (15–64 years old) per day and for ecstasy, approximately 4 doses per 1000 young adults (15–34 years old) per day for ecstasy. [Copyright &y& Elsevier]

Abstract: The present study compares two new sample preparation methods, stir bar sorptive extraction (SBSE) and membrane-assisted solvent extraction (MASE) coupled to the novel technique of ultra-performance liquid chromatography (UPLC) for the sensitive, selective and solvent-free determination of six oxazole fungicide residues (hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl and famoxadone) in wine and juices. The analytes were separated on a rapid resolution C18 column (50mm×4.6mm, I.D., 1.8μm) thermostated at 50°C with isocratic elution using a 50/50 (v/v) water/acetonitrile (ACN) mobile phase at a flow-rate of 1mLmin−1 and detected by diode-array detection (DAD). The UPLC method rapidly separates the fungicides (7min). The best results as regards sensitivity, repeatability and analyte recovery were obtained using SBSE with a polydimethylsiloxane (PDMS) twister, at 60°C for 30min with stirring at 1700rpm in the presence of a 0.1M acetate/acetic acid buffer (pH 5) and 20% (m/v) sodium chloride. Liquid desorption was performed with 100μL of a 80/20 (v/v) ACN/water solution in a desorption time of 15min. With the PDMS polymer, an apolar phase, hymexazol and oxadixyl were not extracted. Consequently, the SBSE procedure can only be applied to the other four fungicides. Detection limits ranged from 0.05 to 2.5μgL−1 at a signal to noise ratio of 3, depending on the compound. Recoveries obtained for spiked samples were satisfactory (83–113%) for all compounds. The proposed method was successfully applied to the analysis of different samples, residues of chlozolinate and drazoxolon being found in samples of red wine and grape juice, respectively. [Copyright &y& Elsevier]

Abstract: A method based on ultra-performance liquid chromatography–electrospray ionisation–tandem mass spectrometry for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with the Decision 2002/657/EC. Samples are extracted with methanol and the extracts cleaned-up on silica cartridges. The recoveries range from 40% for 6-phenyl-2-thiouracil to 79% for 2-thiouracil. Quantification is carried out with blank tissue samples spiked with the analytes in the range 25–500μgkg−1. 5,6-Dimethyl-2-thiouracil is used as internal standard. CCα and CCβ are in the ranges 4.3–16.1μgkg−1 and 8.7–20.7μgkg−1, respectively. Accuracy, expressed as percentage of error, is lower than 6% and relative standard deviation in reproducibility conditions falls between 5.6 and 10.3%. Nowadays, the proposed method is routinely implemented in the laboratory of the Agència de Salut Pública de Barcelona and allows processing of up to 20 samples per day. [Copyright &y& Elsevier]

Abstract: Given that numerous small molecule applications of ultra-performance liquid chromatography (UPLC) have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of 10 protein standards, the optimized method yielded improved chromatographic resolution, enhanced sensitivity, and a threefold increase in throughput. Subsequent cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality at 1/3 of the original run time. [Copyright &y& Elsevier]

Abstract: Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1mm I.D. columns packed with a porous 1.7μm C18 sorbent is below 0.2mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results. [Copyright &y& Elsevier]

Abstract: Liquid chromatography linked to tandem mass spectrometry (LC–MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages since its introduction to the pharmaceutical industry. This article reviews the most recent advances in sample preparation, separation, and the mass spectrometric aspects of high-throughput quantitative bioanalysis of drug and metabolites in biological matrices. Newly introduced techniques such as ultra-performance liquid chromatography with small particles (sub-2μm) and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC–MS/MS. Sample preparation formatted to 96-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly impacting throughput. On-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both time and labor required to produce bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media including large particles for turbulent flow chromatography, restricted access materials (RAM), monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland systems. In the end, this paper also discusses recent studies of matrix effect in LC–MS/MS analysis and how to reduce/eliminate matrix effect in method development and validation. [Copyright &y& Elsevier]

Abstract: An ultra-performance liquid chromatographic (UPLC) electrospray ionisation tandem quadrupole mass spectrometric method has been developed for the determination of 52 pesticides in cereal-based baby foods, oranges and potatoes. The fast polarity switching capability of the mass spectrometer used enabled the determination of 44 of the compounds in the positive ionisation mode and 8 of the compounds in the negative ionisation mode in a single run. Prior to analysis, co-extractives were removed from acetonitrile extracts using dispersive solid-phase extraction with primary secondary amine (50mg). The UPLC method separates all of the pesticides, resolves structural isomers (e.g. butocarboxim sulfoxide and aldicarb sulfoxide) and has a short (7min) cycle time. Extracts spiked with pesticides at 0.10 and 0.01mgkg−1 yielded average recoveries in the range of 66–124% with relative standard deviations less than 19% for the majority of the analytes. [Copyright &y& Elsevier]