Pantothenic acid (vitamin B5) in fortified foods: comparison of a novel ultra-performance liquid chromatography-tandem mass spectrometry method and a microbiological assay (AOAC Official Method 992.07)

A novel method was developed and single-laboratory validated for the determination of free pantothenic acid (vitamin B5) in a wide range of infant and adult fortified food products. The method combines simple sample preparation and chromatographic analysis using ultra-performance LC coupled to tandem MS with positive electrospray ionization. Pantothenic acid was quantified using [13C6, 15N2]-pantothenic acid as an internal standard. Calibration curves were linear between 0.08 and 1.2 microg/mL (r2 = 0.9998), and average recovery varied between 95 and 106%. The method exhibited overall RSD(r) of 1.1% and RSD intermediate reproducibility from 2.5 to 6.0% in infant formulas and cereals. Comparison of results between total and free pantothenic acid showed that the analysis of free pantothenic acid gave a good estimation of total pantothenic acid in the range of products analyzed. The method provides reliable free pantothenic acid results in a wide range of fortified foods (infant and adult nutritionals, cereal products and beverages), and shows good correlation with the microbiological method AOAC Official Method 992.07. It is a more selective, faster, and robust alternative to microbiological determination.     

One saccharide sensor based on the complex of the boronic acid and the monosaccharide using electrochemical impedance spectroscopy

A novel saccharide sensor based on the covalent interaction between the boronic acid and saccharides was developed. Poly (aminophenylboronic acid) (PABA) was prepared by electropolymerizing 3-aminophenylboronic acid on gold electrode surface in acidic solution. The boronic acid group of the PABA film can form covalent-bond with different saccharides, which can change the dielectric characteristics of the PABA film, and the change of the dielectric characteristic was saccharides concentration dependent. Four kinds of saccharides could be detected by using electrochemical impedance spectroscopy. Good linear relationship and high sensitivity were obtained by this method.     

Copolyesteramides: synthesis and kinetic analysis

The synthesis of copolyesteramides using para-acetamido benzoic acid (PABA) and polyethylene terephthalate (PET) by melt polymerization has been studied in detail. The performance of three transesterification catalysts are assessed for three different initial compositions, PABA 60 mol%/PET 40%, PABA 50 mol%/PET 50% and PABA 40 mol%/PET 60%. The polycondensations are found to obey second order kinetics, irrespective of whether the reaction was catalysed or not. The mechanism of initial stage polymerization kinetics of the copolymers has been fully explained.It is suggested that acetic acid is evolved only by the homopolymerization of PABA and that the insertion of a monomer of homopolymer of PABA into PET does not yield any acetic acid. A set of differential equations containing three different rate constants, k₁ for homopolymerization of PABA, k₂ for PET reaction with dimer of PABA and k₃ for PABA reaction with copolymer of PABA and PET has been developed and numerically solved, to study the initial stage kinetics. The computed values of acetic acid are compared with the experimentally collected amount and the three rate constants are optimized using a differential algebraic optimization technique. The present model represents the data with an acceptable accuracy with an average % error of less than 5% between experimental and computed values for the entire experimental range. The correlation coefficient values range between 0.988 and 0.999.Differential scanning analysis of the copolyestermides indicates that 40 mol% PABA and 60% PET had the highest enthalpy values of the order of 18 kJ/mol. It is found that within the copolyesteramide series the degree of crystallinity increased with the increase of PET contents in the feed mixture to the batch reactor.     

Quantitative determination of noncovalently bound acridinium in protein conjugates by liquid chromatography/electrospray ion trap mass spectrometry

A sensitive and robust liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of noncovalently bound acridinium free acid in protein-acridinium conjugates. The lower level of quantitation (LOQ) for acridinium free acid was determined to be 0.6 ng. The assay was validated with a linear concentration range of 0.6-60 ng. The method requires minimum sample handling and is specific, reproducible, and provides a new aspect for protein-acridinium conjugate characterization.     

Sample preparation and gas chromatography of primary fatty acid amides

A method for the isolation of bio-active primary fatty acid amides (PFAM's) from total lipid extract by solid-phase extraction (SPE) was developed and validated. The lowest mass of amide to be loaded and recovered by this method was detected as 0.5 microg using 500 mg of normal phase adsorbent. The isolated PFAM's were separated and quantified by GC/MS and percent recoveries were calculated. An HP-5MS column was able to provide base line separation between the saturated and unsaturated PFAM's whereas clear resolution between geometric and positional isomers having the same number of carbons was obtained using a BPX70 column. The separated amides were all 18 carbon analogs of cis-9-octadecenoamide (oleamide). Detection limits in the single ion monitoring mode were found to be on the order of 10 pg in a 1 microl injection. Solid phase extraction of amides from total lipid extract before GC/MS analysis provides clean detection and interference free analysis.     

Novel magnetic and fluorescent nanocomposite as a sensitive probe for the determination of proteins

The Fe(3)O(4)/(sodium oleic acid/ethyltrimethyl ammonium bromide)(n)/4-aminobenzoic acid (Fe(3)O(4)/(NaOL/CTAB)(n)/PABA) nanocomposites have been prepared by a layer-by-layer self-assembly approach. This kind of nanocomposites have fluorescent, magnetic and water-soluble properties. Taking advantage of the magnetic property of nanocomposites, we can separated them from solution easily by using a permanent magnet. By using their strong fluorescence, we can detect proteins. At pH 6.98, the fluorescence of Fe(3)O(4)/(NaOL/CTAB)(n)/PABA nanocomposites can be enhanced by the proteins. Under optimal conditions, the linear ranges of calibration curves were 0.2-20, 0.2-13, 0.2-10 microg mL(-1) for gamma-globulin (gamma-IgG), human serum albumin (HSA), and bovine serum albumin (BSA), respectively. The detection limits were 0.02, 0.01, 0.02 for gamma-IgG, HSA and BSA, respectively. The method has been applied to analyze the total proteins in human samples and the results were in good agreement with those reported by the hospital. This method is sensitive, simple and potential in many areas.     

液相色谱-四极杆飞行时间质谱法快速测定草莓中6种植物生长调节剂的残留量

建立了高效液相色谱-四极杆飞行时间串联质谱(HPLC-Q TOF MS)快速检测草莓中2,4-滴、对氯苯氧乙酸、3-吲哚丁酸、氯吡脲、脱落酸、玉米素6种植物生长调节剂的分析方法。样品采用QuEChERS方法(Quick, Easy, Cheap, Effective, Rugged and Safe method)进行前处理(乙腈提取,C18吸附剂净化),采用Eclipse XDB-C8色谱柱(150 mm×4.6 mm, 5 μm),以乙腈-5 mmol/L乙酸铵-0.1%(v/v)甲酸水溶液为流动相,梯度洗脱,Q TOF MS电喷雾负离子模式分析检测。在全扫描采集模式下,以准分子离子峰的峰面积定量,以化合物的精确质量数定性。在Target MS/MS采集模式下,通过碎片离子的精确质量数进一步确证化合物。各化合物在0.005～1.0 mg/L范围内均呈现良好的线性关系,相关系数均大于0.99。6种化合物的检出限为1～5 μg/kg,添加回收率为87%～107%,相对标准偏差(RSD)均小于10%(n=6)。本方法简便快捷,选择性好,灵敏度高,可满足国内外现行法规的限量要求。     

GC-MS analysis of organic compounds in wastewater and sewage sludge

A multimethod based on liquid-liquid extraction and solid-liquid extraction for the analysis of persistent organic pollutants in water and sludge from sewage treatment plants has been established. Traces of 22 organic compounds used in industry and personal care products (PCPs) were analyzed by GC/MS. The LODs for the analytes were less than 2.3 ng/L for wastewater and 31 microg/kg (dry weight matter) for sewage sludge. Satisfactory recoveries (70-130%) were achieved. The validated method permits the analysis of water and sludge samples at various stages of the treatment from different sewage treatment plants. Thus, the distribution between water and sludge as well as the dissipation of the compounds analyzed were balanced. By this means, the efficiency of different wastewater treatment plants (WWTPs) can be evaluated and measures can be taken to optimize the treatment process at different stages.     

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Folic acid (FA) plays a vital role in central metabolism, including the one carbon cycle, nucleotide, and amino acid biosynthesis. The development of sensitive, accurate analytical methods to measure FA intermediates in tissues is critical to understand their biological roles in diverse physiological and pathological contexts. Here, we developed a highly sensitive method for the simultaneous quantification of FA intermediates in the nematode Caenorhabditis elegans as a model to dissect metabolic networks. The method was further validated by analyzing the worm folate pool upon RNAi knockdown of the dihydrofolate reductase gene dhfr‐1. Comparative mass spectrometry behavior of the FA analogs using two different ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), revealed ESI‐MS/MS to be more sensitive, but APCI‐MS provided more detailed structure inferences, which can elucidate chemical investigation and synthesis of FA analogs. Finally, we report on the use of in vitro oxidation coupled with high‐resolution mass spectrometry as a tool to discover new endogenous FA derivatives in the nematode.     

Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi (Radix astragali) by HPLC-DAD-ELSD

A simple, rapid, and reliable method, namely high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD), was developed to simultaneously determine twelve major flavonoids and five main saponins in different parts of the medicinal plant Huang-qi (Radix Astragali). The DAD wavelength was set at 280 nm for the UV detection of flavonoids, while the drift tube temperature was set at 105 degrees C and the nebulizing gas flow rate at 2.7 L/min for ELSD detection of saponins. The method was fully validated with respect to linearity (r2 >0.998), sensitivity, precision, and accuracy (recovery rate between 93.3 and 104.2%). The analytical results of different parts of the medicinal plant Huang-qi revealed that the levels of total flavonoids or saponins in individual parts can vary considerably and the concentration of each compound in different parts is also significantly different. The aerial parts (stems and leaves) contain even higher total contents of flavonoids (although of different kinds) than the commonly used roots of the plants. In addition, the concentration of total flavonoids and saponins in the extract of the fibrous roots was surprisingly highest among all parts of Astragalus species. All of these findings provide clear evidence and scientific support for utilization of different parts of the medicinal plant Huang-qi and also for reduction in waste of plant resources.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.     

Separation and Determination of Chemopreventive Phytochemicals of Flavonoids from Brassicaceae Plants

The main aim of this study was to develop a method for the isolation and determination of polyphenols—in particular, flavonoids present in various morphological parts of plants belonging to the cabbage family (Brassicaceae). Therefore, a procedure consisting of maceration, acid hydrolysis and measurement of the total antioxidant capacity of plant extracts (using DPPH assay) was conducted. Qualitative analysis was performed employing thin-layer chromatography (TLC), which was presented to be a suitable methodology for the separation and determination of chemopreventive phytochemicals from plants belonging to the cabbage family. The study involved the analysis of 25 vegetal samples, including radish, broccoli, Brussels sprouts, kale, canola, kohlrabi, cabbage, Chinese cabbage, red cabbage, pak choi and cauliflower. In addition, selected flavonoids content in free form and bonded to glycosides was determined by using an RP-UHPLC-ESI-MS/MS method.     

The effect of water on the ion trap analysis of trimethylsilyl derivatives of long-chain fatty acids and alcohols

Gas chromatography/mass spectrometry (GC/MS), with an ion trap mass analyzer, was used to examine the very-long-chain cuticular acid and certain non-acid wax constituents on the leaf sheath surface of Sorghum bicolor before and during 36 hours of light exposure. The mass spectra of the trimethylsilylated acids and alcohols did not match any of those published in searchable mass spectral libraries. The observed differences can be related to the interaction between water and the trimethylsilylated acids and alcohols. Understanding the observed mass spectra of the very-long-chain plant waxes is critical for studies that employ GC/MS with the ion trap mass analyzer to elucidate cuticular wax compositions on plants.     

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.     

Application of an LC–MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC–MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa’s k = 0.6) and a strong correlation (Pearson’s r = 0.73). To our knowledge, this is the first study to utilize LC–MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.     

Metabolomics analysis reveals variation in Schisandra chinensis metabolites from different origins

Wu Wei Zi (Schisandra chinensis), an important herbal medicine, is mainly distributed in the northeast of China. Its phytochemical compositions, which depend on geographical origin, climatic conditions and cultural practices, may vary largely among Wu Wei Zi from different areas. In this study, we applied a comprehensive metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC (UPLC) quadrupole TOF (QTOF) MS and inductively coupled plasma MS to systematically investigate the metabolite variations of S. chinensis from four different areas including Heilongjiang, Liaoning, Jilin, and Shanxi of China. A total of 65 primary metabolites, 35 secondary metabolites and 64 inorganic elements were identified. Several primary metabolites, including shikimic acid and tricarboxylic acid cycle intermediates, were abundant in those located in Heilongjiang, Jilin, and Liaoning. Besides, bioactive lignans are also highly abundant in those from northeastern China than those from northwestern China. Inorganic elements varied significantly among the different locations. Our results suggested that the metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC quadrupole TOF MS, and inductively coupled plasma MS is a robust and reliable method that can be effectively used to explore subtle variations among plants from different geographical locations.     

Determination of para-aminobenzoic acid,a degradation product of procaine hydrochloride,by zero-crossing first-derivative spectrometry

A first derivative spectrometric method has been developed for the determination of the para-aminobenzoic acid (PABA), a related substance and degradation product of the local anesthetic procaine hydrochloride (PH). The wavelength selected for the determination of para-aminobenzoic acid in the presence of procaine was 290 nm. At this wavelength the calibration graphs relating the amplitude of the signals to the concentration of para-aminobenzoic acid were linear up to 10 mg L(-1). The detection limit of para-aminobenzoic acid was 0.70 mg L(-1). The method is simple and rapid and does not require preliminary treatment of the sample. The method was validated.     

Identification and quantification of flavonoids and chromes in Baeckea frutescens by using HPLC coupled with diode-array detection and quadruple time-of-flight mass spectrometry

This article marks the first report on high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and quadruple time-of-flight mass spectrometry (Q-TOF/MS) for the identification and quantification of main bioactive constituents in Baeckea frutescens. In total, 24 compounds were identified or tentatively characterised based on their retention behaviours, UV profiles and MS fragment information. Furthermore, a validated method with good linearity, sensitivity, precision, stability, repeatability and accuracy was successfully applied for simultaneous determination of five flavonoids and one chromone in different plant parts of B. frutescens collected at different harvest times, and their dynamic contents revealed the appropriate harvest times. The established HPLC-DAD-Q-TOF/MS using multi-bioactive markers was proved to be a validated strategy for the quality evaluation on both raw materials and related products of B. frutescens.     

LC–MS/MS method for the characterization of the forced degradation products of Entecavir

A rapid, specific, and reliable isocratic LC–MS/MS method has been developed and validated for the identification and characterization of the stressed degradation products of Entecavir (ETV). ETV, an antiviral drug, was subjected to hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis and thermal stress, as per the international conference on harmonization specified conditions. The drug showed extensive degradation under oxidative and acid hydrolysis stress conditions. However, it was stable to thermal, acidic, neutral, and photolysis stress conditions. A total of five degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on a Waters Symmetry C₁₈ (250 mm × 4.6 mm, id, 5 μm) column using 20 mM ammonium acetate (pH 3)/acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. The LC–MS method was validated with respect to specificity, linearity, accuracy, and precision. No previous reports were found in the literature regarding the degradation behavior of ETV.     

Analyses of chlorogenic acids and related cinnamic acid derivatives from <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method

Background

Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures.

Results

Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof.

Conclusion

Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.