Positive Atmospheric Pressure Chemical Ionization Research Articles

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca

Ethnopharmacological relevanceSnakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. Aim of the studyTo investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. Materials and methodsThe ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. ResultsA total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70–90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. ConclusionsAltogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Development of an analytical method for the determination and quantification of n-nitrosodimethylamine in olmesartan by HPLC-MS/MS

In this study, a simple, rapid, and highly sensitive analytical method has been developed and validated for the detection, separation, and quantification of N-nitrosodimethylamine in the active pharmaceutical ingredient olmesartan medoxomil, as well as in manufactured tablets and marketed tablets, using high-performance liquid chromatography-mass spectrometry. Chromatographic separation was achieved using the Agilent Eclipse XDB-C18 column (5 m × 4.6 mm × 150 mm) with a binary gradient with water as mobile phase A and 0.1 % formic acid in methanol as mobile phase B, flow rate 0.5 mL/min, B. Due to the presence of polar groups, N-nitrosodimethylamine is successfully ionized and quantified in positive atmospheric pressure chemical ionization mode using multiple reaction monitoring. The method was validated according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines, evaluating the limit of detection and quantification, a good signal-to-noise ratio, and an excellent regression coefficient greater than 0.995 were found. The accuracy (expressed as recovery) of the method was performed in triplicate obtaining satisfactory results between 80 and 104 %, with a precision (%RSD) of 0.34 – 1.50 %. The method can be used routinely for the determination and quantification of N-nitrosodimethylamine in olmesartan.

Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS.

Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Steroids Androstenedione, Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone.

Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive disorders due to deficiency of 11- or 21-hydroxylase. The analysis of cortisol, androstenedione, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Analysis of specific steroids is also performed in other disorders such as evaluation of hirsutism or infertility in females and hypogonadism in males. Cortisol is generally analyzed by immunoassays, whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC-MS/MS method is described for the simultaneous quantification of androstenedione, 17-hydroxyprogesterone, DHEA, 11-deoxycortisol and testosterone. The method involves addition of labeled internal standards to serum samples and extraction of steroids in methyl tert-butyl ether. The extract is evaporated under stream of nitrogen, and the residue is reconstituted in methanol and analyzed by LC-MS/MS.

Oxidation of diethyl ether: Extensive characterization of products formed at low temperature using high resolution mass spectrometry

The oxidation of a stoichiometric diethyl ether-oxygen-nitrogen mixture containing 5000 ppm of fuel was studied in a jet-stirred reactor at 10 atm and a residence times of 1 s. Experimental temperatures varied stepwise for 440 to 740 K and products were quantified by gas chromatography with TCD and FID, gas chromatography-mass spectrometry, and FTIR. Other experiments in the temperature range 480 to 570 K were performed for characterizing elusive cool flame products. To this end, gas samples were trapped in acetonitrile for flow injection analyses and liquid chromatography-mass spectrometry (Orbitrap Q-ExactiveⓇ). For ionization, we used positive and negative atmospheric pressure chemical ionization (APCI). Among fuel-specific products, hydroperoxides and diols (C4H10O3), carbonyl hydroperoxides (C4H8O4), acetic acid, di-keto ethers (C4H6O3), cyclic ethers (C4H8O2) and highly oxygenated molecules, i.e., keto-dihydroperoxides (C4H8O6), keto-trihydroperoxides (C4H8O8), di-keto-hydroperoxides (C4H6O5), and diketo-dihydroperoxides (C4H6O7), were detected. To confirm the presence of –OH or –OOH groups in oxidation products, H/D exchange with D2O was used. DNPH derivatization was used to identify carbonyls present in samples, especially those with a molecular weight below 50 amu which cannot be detected directly by the mass spectrometer. Chemical kinetic modeling using a mechanism taken from the literature was performed. Although reasonable agreement between the data and the simulations was observed for several species, some discrepancies between experimental and computed mole fractions were observed.

Research progress on analytical methods for the determination of hexachlorobutadiene

六氯丁二烯是一种持久性有机污染物,于2015年和2017年分别被列入《斯德哥尔摩公约》附件A和附件C的受控污染物名单中。六氯丁二烯的来源、环境赋存和影响等研究对控制该新增受控持久性有机污染物污染具有重要意义,而灵敏可靠的六氯丁二烯分析方法是开展相关研究的前提和基础。近年来已有不少学者将六氯丁二烯作为分析目标物之一进行了检测或方法学研究。基于这些研究成果,该文综述了六氯丁二烯分析方法的研究进展,其中重点介绍了空气、水体、土壤、污泥、生物组织等多种介质中六氯丁二烯的样品前处理方法,并比较了各方法的优缺点,以期为该领域的进一步研究提供参考。空气中六氯丁二烯主要由泵抽气通过吸附管而采集,再经热脱附后进行仪器分析,检出限在ng/m3水平。也有研究应用聚氨酯泡沫被动采样器和吸附剂填充聚氨酯泡沫被动采样器采集大气中六氯丁二烯及其他污染物。基于吸附剂填充聚氨酯泡沫被动采样器的分析方法灵敏度较高,其对六氯丁二烯的检出限低至0.03 pg/m3。然而目前被动采样体积仅根据六氯丁二烯的log KOA系数估算,未来仍需进一步实验校正。水体样品前处理通常也较简单,通过吹扫捕集、液-液萃取或固相萃取目标物后进行仪器分析。固相萃取法能够同步实现目标物的提取、净化和浓缩,在水样中六氯丁二烯分析方面具有明显优势。固相萃取柱类型以及干燥步骤中柱中残留水分去除率均会影响六氯丁二烯的回收率。灰尘、土壤、沉积物、污泥和生物组织等固体介质样品基质最为复杂,需联合多种方法进行前处理。固体样品中六氯丁二烯提取方法包括索氏提取,加速溶剂萃取和超声萃取,其中超声萃取法应用最为广泛。固体基质净化方面主要采用层析柱色谱法,多根净化柱联用或多层复合柱能够提升净化效果。仪器分析方面,六氯丁二烯主要采用气相色谱和质谱联用检测,高性能质谱检测器如串联质谱能够大大提高六氯丁二烯的检测灵敏度,具有较大的应用潜力。

SAT-LB13 Clinical Utility of 21-Deoxycortisol in Congenital Adrenal Hyperplasia

Introduction Congenital Adrenal Hyperplasia (CAH) is most often caused by mutation of the 21-hydroxylase gene (CYP21), which results in underproduction of cortisol with overproduction of precursor steroids and their metabolites by the adrenal glands. Historically the most common biomarker used for detecting CAH in pediatric patients is 17-Hydroxyprogesterone (17OHP). Another less commonly used biomarker for 21-Hydroxylase deficiency is 21-deoxycortisol (21DOF), which increases from very low levels in normal patients to high levels in affected patients as 17OHP rises to very high levels. In this study we performed retrospective analysis of serum 21DOF concentration in specimens that had been genotyped for mutations in the CYP21A2 gene, or had been submitted to our laboratory for provocative adrenocorticotropin (ACTH / Cosyntropin) stimulation testing. Methods: Biochemical testing for 21DOF concentration was measured by LC-MS/MS. Briefly, a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1100 pumps (Agilent Technologies, Inc.) and a Sciex® 5000 (Danaher) triple quadrupole mass spectrometer in positive mode atmospheric pressure chemical ionization (APCI) was used for detection in Multiple Reaction Monitoring (MRM) mode. Genetic testing was performed using the CAHDetx test, which detects the 12 most common small mutations and large gene deletions/conversions in CYP21A2. Genetic Correlations: 21DOF was quantifiable (above the LLOQ of the assay) in 4% (n=24/600) of specimens where no mutation was detected. 21-DOF was quantifiable in 42% (122/292) of specimens with 21-hydroxylase enzyme mutations as determined by the CAHDetx test. Those mutations included In2G, I172N, V281L and others. Some mutations such as Q318X did not result in a detectable increase in 21-deoxycortisol. ACTH Stimulation Response: 21-deoxycortisol was below the quantitation limit in both the baseline and stimulated samples in ~35% (52/148) of submitted samples. The 21-deoxycortisol was quantifiable in only the stimulation sample in ~45% (65/148) of ACTH stimulation submitted, and was quantifiable in both baseline and stimulated samples in the remaining ~20% (30/148) of ACTH stimulation pairings. The extent of 21-deoxycortisol increase ranged from 1.2-fold to 116-fold with a median 14-fold increase. Clinical Significance: The use of 21-deoxycortisol may be beneficial in reducing the rate of false positives in CAH diagnosis when used in concert with other steroid hormones, and may eventually reduce the need for provocative testing to confirm CAH diagnosis.

Reversed phase UPLC/APCI-MS determination of Vitamin K1 and menaquinone-4 in human plasma: Application to a clinical study

A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K1 (PK) and vitamin K2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE –PFP C18 column (50 × 2.1 mm, 3 μ) equipped with standard C18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5 mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01 ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01–50 ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81 ng/mL and 0.33–0.57 ng/mL, respectively.

Ionization mechanism in gel permeation chromatography mass spectrometry (GPC-MS) with atmospheric pressure chemical ionization (APCI) interface

The ionization mechanism in gel permeation chromatography (GPC) coupled with mass spectrometry (MS) using an atmospheric pressure chemical ionization (APCI) interface is explained when chloroform is used as GPC solvent. The even electron anions [CHCl3+Cl]− generated by thermal electrons in negative APCI mode are responsible for further ionization of the solute compounds as [M + Cl]− adducts. In positive APCI mode, the contaminants in chloroform generate a relatively high background noise. These cations can be suppressed by adding 10% of tetrahydrofuran (THF). Supporting the described mechanism are key experiments done in the absence of solvent, in GPC solvents and using a mixture of anionic polystyrene (PS) standards from dimer up to 12,000 g/mol, covering the molecular masses of great interest in studies related to Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) and Food and Drug Administration (FDA). Advantages of suppressing the oligomer signals for polymer additive analyses were presented using Irgafos® 168.

Development and validation an LC-MS/MS method to quantify (+)-borneol in rat plasma: Application to a pharmacokinetic study

(+)-Borneol, a bicyclic monoterpene, has been shown to possess valuable biological properties and potential as a pharmaceutical agent due to anti-inflammatory, anti-oxidant and GABA receptor-enhancing functions; it also enhances the permeability of the blood brain barrier to improve the efficacy of CNS drugs. In this study, we have developed a simple, selective, and rapid liquid chromatography-tandem mass spectrometry method for the assay of (+)-borneol in rat plasma. Verapamil was used as an internal standard. Plasma samples were deproteinized using methanol. The analyte was detected by a mass spectrometer with positive atmospheric pressure chemical ionization by multiple reaction monitoring mode for transitions at m/z [M + H]+ 137.2 → 81.0 for (+)-borneol and 455.2 → 165.1 for verapamil. The method has been fully validated to ensure good selectivity, a satisfactory lower limit of quantification at 10.0 ng/mL, acceptable intra- and inter-day accuracy, and high precision. The method was used for the pharmacokinetic evaluation of (+)-borneol in Sprague-Dawley rats after intravenous, oral, and sublingual administration. The results indicate that oral bioavailability of (+)-borneol was extremely low but sublingual administration yielded rapid absorption and favorable bioavailability of (+)-borneol.

Profiling of Hura crepitans L. latex by ultra-high-performance liquid chromatography/atmospheric pressure chemical ionisation linear ion trap Orbitrap mass spectrometry.

The phytochemistry of the latex of Hura crepitans L. (Euphorbiaceae), a widespread tree in the Amazonian forest having many uses, is little known. Only huratoxin, a daphnane diterpene orthoester, has been described despite the high pharmacological potential of this kind of compounds. Glucosphingolipids (cerebrosides) are also known to be distributed in Euphorbiaceae latexes. To tentatively identify daphnanes diterpenes and cerebrosides in the latex of H. crepitans. An ethanolic extract of the lyophilised latex of H. crepitans was analysed by ultra-high-performance liquid chromatography (UHPLC) coupled with positive and negative atmospheric pressure chemical ionisation high-resolution mass spectrometry (APCI-HRMS) method using a quadrupole/linear ion trap/Orbitrap (LTQ-Orbitrap). Tandem mass spectrometry (MS/MS) spectra were recorded by two different fragmentation modes: collision induced dissociation (CID) and higher-energy collisional dissociation (HCD). The analysis of CID- and HCD-MS/MS spectra allowed to propose fragmentation patterns for daphnane esters and cerebrosides and highlight diagnostic ions in positive and negative ion modes. A total of 34 compounds including 24 daphnane esters and 10 cerebrosides have been tentatively annotated. Among them, 17 daphnane diterpenes bearing one or two acyl chains are new compounds and the cerebrosides are described in the genus Hura for the first time. This study revealed the chemical constituents of the latex of H. crepitans and particularly its richness and chemical diversity in daphnane diterpenes, more frequently encountered in the species of Thymelaeaceae.

High-performance liquid chromatography quantification of enantiomers of a Dihydroxylated tetrahydrofuran natural product.

Both enantiomers of petromyroxol are putative pheromones in sea lamprey (Petromyzon marinus). Here, we describe the separation and quantification of the petromyroxol enantiomers using high-performance liquid chromatography tandem mass spectrometry. The separation was tested on a wide range of chiral columns with normal phases, and effects of the chromatographic parameters such as mobile phase and temperature on the separation were optimized. The AD-H column showed the best separation of enantiomers with n-hexane and ethanol as the mobile phase. The enantiomers were detected by multiple reaction monitoring with a positive atmospheric-pressure chemical ionization on triple quadrupole mass spectrometer. Validation revealed that the method was specific, accurate, and precise. The validated method was applied to measure the amount of petromyroxol enantiomers in water conditioned with sea lamprey larvae, the source of the putative pheromone. This method will be applied in quantifying the natural scalemic petromyroxol mixture, enabling further investigations of a rare non-racemic enantiomeric pheromone mixture in a vertebrate species.

Application of supercritical fluid chromatography coupled to mass spectrometry to the determination of fat-soluble vitamins in selected food products

In the present manuscript, we describe a fully optimized and validated method suitable to analyse nine compounds (retinyl acetate, retinyl palmitate, retinol, α-tocopherol, α-tocopheryl acetate, cholecalciferol, ergocalciferol, phylloquinone, menaquinone-4) representing the major contributors to the fat-soluble vitamin activity of selected food products (infant formulas, adult nutritionals, infant cereals and mixed meals). Sample preparation involves direct solvent extraction using enzyme-assisted matrix disintegration and methanolic protein precipitation. Direct injection of the extract allows quantification of vitamins A, E and K in only 7 min, while vitamin D is determined after fast derivatization of the extract. Separation is achieved by supercritical fluid chromatography and detection performed by tandem mass spectrometry in positive Atmospheric Pressure Chemical Ionization mode. Results on a Standard Reference Material (SRM 1849a Infant/Adult Nutritional) were not statistically different from reference values. Full validation of the method showed excellent overall performance. Average recovery rate was between 90 and 110% for all vitamins and matrixes. The methodology shows enhanced safety and reduced cost as compared with previously published methods, together with potential for application to more complex matrixes. The full procedure can be easily applied in control laboratories dramatically increasing sample throughput and reducing solvent consumption.

Coupling supercritical fluid chromatography to positive ion atmospheric pressure ionization mass spectrometry: Ionization optimization of halogenated environmental contaminants

Currently used analytical techniques for halogenated aromatic environmental contaminants such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs), also known as legacy persistent organic pollutants, are based on gas chromatographic separation of target analytes and detection by mass spectrometry. The coupling of packed column supercritical fluid chromatography (SFC) to atmospheric pressure ionization mass spectrometry (API/MS) could allow for the concurrent analysis of thermally labile and legacy halogenated environmental contaminants if ionization can be sufficiently optimized. The evaluation of positive ion atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) as well as possible charge transfer dopants for the generation of molecular ion isotopomeric clusters of halogenated environmental contaminants with minimal fragmentation has been completed. Using the investigated parameters, positive ion APPI was found to be the more sensitive technique. Of the aromatic and cycloalkane dopants investigated, only fluorobenzene and trifluorotoluene were found to be effective dopants for the halogenated aromatic target analytes (PCDDs, PCDFs, and PCBs). Experiments involving deuterated dopants confirmed that reactive species generated by cycloalkanes were quenched by the SFC eluent rendering them unusable in conjunction with the investigated separation technique. Alternatively, aromatic dopants were found to be less susceptible to quenching by the SFC eluent and fluorobenzene was determined to be the most effective charge transfer dopant for PCDDs, PCDFs, and PCBs. To demonstrate the applicability of the optimized ionization conditions, SFC-API/MS has been used for the concurrent analysis of legacy halogenated aromatic environmental contaminants (PCDDs, PCDFs, and PCBs) and thermally labile analytes (α, β, and γ isomers of hexabromocyclododecane).

Isotope Dilution UPLC-APCI-MS/MS Method for the Quantitative Measurement of Aromatic Diamines in Human Urine: Biomarkers of Diisocyanate Exposure.

Urinary diamines are biomarkers of diisocyanate exposure. Diisocyanates are considered as skin and respiratory sensitizers and are the most frequently reported cause of occupational asthma. Herein we report on the development and validation of an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of five aromatic diamines, 4,4'-methylenedianiline (MDA), 2,4-toluenediamine (4TDA), 2,6-toluenediamine (6TDA), 1,5-naphthalenediamine (NDA), and p-phenylenediamine (PPDA) in human urine. The method incorporates sample preparation steps, which include a 4 h acid hydrolysis followed by high-throughput solid-phase extraction prior to chromatographic separation. Chromatographic separation was achieved using a C18 reversed phase column with gradient elution of basic mobile phases (pH 9.2). The duty cycle of the method was less than 5 min, including both the column equilibration and autosampler movement. Analytical detection was performed using positive ion atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) in scheduled multiple reaction monitoring (sMRM) mode. Excellent linearity was observed over standard calibration curve concentration ranges of 3 orders of magnitude with method detection limit ranging from 10 to 100 pg/mL. The interday and intraday reproducibility and accuracy were within ±15%. This method is fast, accurate, and reproducible and is suitable for assessment of exposure to the most common aromatic diisocyanates within targeted groups as well as larger population studies such as the National Health and Nutrition Examination Survey (NHANES).

Simultaneous determination of β-sitosterol, campesterol, and stigmasterol in rat plasma by using LC-APCI-MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of β-sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol-d6 was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 μL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 μL) samples were extracted by liquid-liquid extraction with methyl tert-butyl ether and separated on a Kinetex C18 column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250-5000 ng/mL (β-sitosterol), 250-5000 ng/mL (campesterol), and 50-2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.

Quantitative profiling of retinyl esters in milk from different ruminant species by using high performance liquid chromatography-diode array detection-tandem mass spectrometry

An effective high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) analytical approach was developed for retinoid profiling in raw milk samples (cow, buffalo, ewe, and goat). The analytes were isolated by means of liquid-liquid extraction, including a “lipid freezing” step, with yields exceeding 66%. Since the positive atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) detection is not completely selective, a reliable identification has been accomplished by fully separating the analytes on a tandem C18/C30 column system under non-aqueous reversed phase (NARP) chromatography conditions. After validation, different milk varieties obtained from pasture-fed animals were analysed, providing, for the first time, the retinoid composition of both buffalo’s and ewe’s milk. According to the literature, retinyl palmitate has been found to be the most abundant vitamin A vitamer, but retinyl oleate is the prevalent form in the caprine milk.

Liquid chromatography/mass spectrometry identification of intermediates and vulcanization products by using squalene as vulcanization model compound.

Sulfur-vulcanized rubber is a three-dimensional polymer network, insoluble in all organic solvents. For this reason, vulcanization products are difficult to study and identify by conventional analytical techniques. To simplify this task, low molecular weight olefins have been used as model compounds (MCs) in place of rubber in vulcanization experiments. In this work, the vulcanization process was investigated using squalene (SQ) as MC. By-products, intermediates and products were separated by semipreparative reversed-phase liquid chromatography (RPLC) with UV detection. Each fraction was collected, concentrated and characterized by flow injection analysis (FIA) and non-aqueous reversed-phase (NARP) LC coupled to positive atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Under the latter conditions, an Information-Dependent Acquisition (IDA) was performed on a linear ion trap mass spectrometer to obtain structural information. Several vulcanized compounds containing up to three SQ molecules, cross-linked with chains involving up to 14 sulfur atoms overall, have been identified along with some of their oxidized products (epoxides and hydroperoxides). The FIA-MS spectra showed peak clusters, each of which included two-three subclusters; the interpretation was complicated by the occurrence of more ion species per product, by the unsaturation grade and by the characteristic isotopic distribution of sulfur. The enhanced product ion scan (EPI) spectra, acquired during the IDA experiments, supported the FIA-MS identification allowing one to count the number of sulfur atoms. The sensitivity of the developed analytical strategy was due to the enrichment factor achieved via semipreparative chromatography and the very good response of the APCI detection. Pattern fragmentation and chromatographic behavior simplified the identification of the cured compounds and their oxidized products, whose occurrence was related to the grade of oxidation of SQ used as reagent. Copyright © 2016 John Wiley & Sons, Ltd.

Optimization and application of atmospheric pressure chemical and photoionization hydrogen-deuterium exchange mass spectrometry for speciation of oxygen-containing compounds.

This paper presents a detailed investigation of the feasibility of optimized positive and negative atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and atmospheric pressure photoionization (APPI) MS coupled to hydrogen-deuterium exchange (HDX) for structural assignment of diverse oxygen-containing compounds. The important parameters for optimization of HDX MS were characterized. The optimized techniques employed in the positive and negative modes showed satisfactory HDX product ions for the model compounds when dichloromethane and toluene were employed as a co-solvent in APCI- and APPI-HDX, respectively. The evaluation of the mass spectra obtained from 38 oxygen-containing compounds demonstrated that the extent of the HDX of the ions was structure-dependent. The combination of information provided by different ionization techniques could be used for better speciation of oxygen-containing compounds. For example, (+) APPI-HDX is sensitive to compounds with alcohol, ketone, or aldehyde substituents, while (-) APPI-HDX is sensitive to compounds with carboxylic functional groups. In addition, the compounds with alcohol can be distinguished from other compounds by the presence of exchanged peaks. The combined information was applied to study chemical compositions of degraded oils. The HDX pattern, double bond equivalent (DBE) distribution, and previously reported oxidation products were combined to predict structures of the compounds produced from oxidation of oil. Overall, this study shows that APCI- and APPI-HDX MS are useful experimental techniques that can be applied for the structural analysis of oxygen-containing compounds.

High-Throughput Serum 25-Hydroxy Vitamin D Testing with Automated Sample Preparation.

Serum from bar-coded tubes, and then internal standard, are pipetted to 96-well plates with an 8-channel automated liquid handler (ALH). The first precipitation reagent (methanol:ZnSO4) is added and mixed with the 8-channel ALH. A second protein precipitating agent, 1 % formic acid in acetonitrile, is added and mixed with a 96-channel ALH. After a 4-min delay for larger precipitates to settle to the bottom of the plate, the upper 36 % of the precipitate/supernatant mix is transferred with the 96-channel ALH to a Sigma Hybrid SPE(®) plate and vacuumed through for removal of phospholipids and precipitated proteins. The filtrate is collected in a second 96-well plate (collection plate) which is foil-sealed, placed in the autosampler (ALS), and injected into a multiplexed LC-MS/MS system running AB Sciex Cliquid(®) and MPX(®) software. Two Shimadzu LC stacks, with multiplex timing controlled by MPX(®) software, inject alternately to one AB Sciex API-5000 MS/MS using positive atmospheric pressure chemical ionization (APCI) and a 1.87 min water/acetonitrile LC gradient with a 2.1 × 20 mm, 2.7 μm, C18 fused core particle column (Sigma Ascentis Express). LC-MS/MS through put is ~44 samples/h/LC-MS/MS system with dual-LC channel multiplexing. Plate maps are transferred electronically from the ALH and reformatted into LC-MS/MS sample table format using the Data Innovations LLC (DI) Instrument Manager middleware application. Before collection plates are loaded into the ALS, the plate bar code is manually scanned to download the sample table from the DI middleware to the LC-MS/MS. After acquisition-LC-MS/MS data is analyzed with AB Sciex Multiquant(®) software using customized queries, and then results are transferred electronically via a DI interface to the LIS. 2500 samples/day can be extracted by two analysts using four ALHs in 4-6 h. LC-MS/MS analysis of those samples on three dual-channel LC multiplexed LC-MS/MS systems requires 19-21 h and data analysis can be done by two analysts in 4-6 h.