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Various types of milk powder purportedly providing diverse health functions have emerged with the growth of the country's elderly population. Some manufacturers illegally add chemical drugs to their products to achieve their reported benefits, which poses a threat to consumer health. The existing standard methods are inapplicable to such complex sample matrices and require testing based on functional claims and classification. These limitations not only consume manpower and resources but also seriously impede daily regulatory efforts to detect unknown risk substances. In this study, a high-throughput method for the screening and quantitative analysis of 300 illegally added chemical drugs in functional milk powder and an identification strategy for unknown structural analogues were established using Zeno SWATH® data-independent acquisition (DIA) ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) technology combined with a QuEChERS sample purification method. The QuEChERS purification process was developed according to the characteristics of milk powder matrix. The supernatant was separated on a Kinetex F5 column (100 mm×3.0 mm, 2.6 µm) by gradient elution using 5 mmol/L ammonium formate aqueous solution (0.1% (v/v) formic acid, ) and methanol-acetonitrile (1∶1, v/v) as mobile phases. The method was validated in terms of selectivity, linearity, limits of detection and quantification (LODs and LOQs, respectively), matrix effect, accuracy, and precision. Based on a screening database for the 300 target substances, electron-activated dissociation (EAD) fragmentation was applied to obtain rich secondary MS fragmentation information, and unknown structural analogues were identified and confirmed through fragment attribution analysis. The results indicated that all compounds had good linear relationships in certain ranges with correlation coefficients >0.99. The LODs and LOQs were 0.04-2.7 and 0.2-8.0 µg/kg, respectively. The average recoveries at three spiked levels were in the range of 73.1%-125.2%, and the relative standard deviations were ≤14.8% (n=6). When the developed method was applied to detect illegally added chemicals in 60 functional milk powder samples, it detected benzoguanidine and sildenafil and successfully identified ethylphenidate, which is the structural analogue of an amphetamine. The proposed method is simple, sensitive, and accurate; thus, it may have practical application value for the daily supervision and law enforcement of milk powders with reported health functions.

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Dapagliflozin (DAPA), sodium-glucose co-transporter 2 (SGLT-2) inhibitor, is used to treat Type 2 diabetes. In this study, a highly sensitive and selective analytical method based on ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was established and validated for the determination of DAPA in rat plasma. The separation of DAPA and internal standard (DAPA-d5) were performed on a reversed-phase ACQUITY UPLC® BEH C18 column (100 × 3.0 mm, 1.7 µm). The mobile phase is composed of 0.1% formic acid in water (solvent A) and methanol (solvent B) in gradient elution. Under the negative ion mode, full MS/dd-MS2 was adopted to collect data via Q-Orbitrap. DAPA was effectively separated from matrix backgrounds within 10 min, and DAPA in plasma showed a good linear relationship in the range of 10-10000 µg/L. The determination coefficient (R2) was 0.9987, and the lower limit of quantification (LLOQ) was 10 µg/L. The precision and accuracy were all less than 10%, and the extraction recovery of DAPA was 86.16-96.06% from plasma. This study offered an efficient separation and quantification method for DAPA. The improved and validated method succeeded in evaluating the pharmacokinetics of DAPA in rat plasma samples after a single oral administration of 1 mg/kg.

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For confirmation and/or screening purposes, rapid, selective, and precise chromatographic methods are required. In this vein, the utility of SiH columns (C18, UDC Cholesterol, and Diamond Hydride) with photodiode array UV absorption and single quadrupole MS detection for multi-modal separation of representative drugs from different drug classes on a single column using the same solvent reservoirs was investigated. For a conventional two column approach employing a combination of conventional C18 and silica columns operating in both the reversed phase chromatographic and hydrophilic interaction chromatographic modes, gradient analysis is required for the first column and there is a lack of retention on the second column for non-amine analytes. In comparison, all analytes are retained for two relatively rapid (< 10 min), precise (% RSD <0.4%), and non-correlated isocratic separations (R2=0.2115) when using a UDC Cholesterol column.

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Sophorolipids are secondary metabolites produced during fermentation by nonpathogenic yeasts. These molecules are amphiphilic and consist of a hydrophilic sophora sugar moiety and a hydrophobic hydroxylated fatty acid. Based on their degree of esterification, sophorolipids can be divided into the acid and lactone types. Sophorolipids are highly promising biosurfactants with good antibacterial, antiviral, and other biological activities. Moreover, they are characterized by mildness, low toxicity, and environmental friendliness. However, their composition is quite complex, and effective methods for their quality evaluation are lacking. Since sophorolipids do not absorb ultraviolet (UV) light, common UV detectors are unsuitable for fingerprint establishment. In this study, we first selected a charged aerosol detector (CAD) to establish the ultra-high performance liquid chromatography (UHPLC) fingerprint of sophorolipids. The detector had high sensitivity, good reproducibility, and excellent suitability for the detection of substances with no or weak ultraviolet absorption. We then evaluated the similarities between 17 batches of sophorolipid samples. The samples were extracted by ultrasound for 10 min in 80% ethanol aqueous solution at a liquid-solid ratio of 10∶1 (mL/g) and then separated on a Thermo Fisher Scientific Hypersil Gold chromatographic column (150 mm×2.1 mm, 1.9 µm). Separation was performed using acetonitrile-0.01% (v/v) formic acid aqueous solution as the mobile phase via gradient elution. The flow rate was 0.2 mL/min, and the column temperature was 40 ℃. The CAD was used under the following conditions: power function of 1.0, data rate of 5 Hz, filter constant of 3.6, and evaporation temperature of 45 ℃. The chromatograms and retention times of the sophorolipids were compared, and 16 common peaks with strong responses, good resolutions, and stable retention times were selected as characteristic peaks. Oleic acid was chosen as the reference peak because it achieved good separation and a strong chromatographic response in all batches of samples. UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was used to identify chromatographic peaks in the sophorolipid fingerprints. The results were combined with the retention time rule of the sophorolipids, leading to their identification based on matching with the results of the primary database, the precise relative molecular mass and fragmentation rule of secondary fragments, a self-built database, and the PubChem database. Sixteen compounds were identified, including eight acid sophorolipids, six lactone sophorolipids, and two aliphatic acids. The results of precision, repeatability, and 24 h stability tests indicated that the relative standard deviations (RSDs) of the retention times and peak areas of the 15 characteristic peaks relative to the control peak (oleic acid) were less than 3.0% (n=6). Seventeen batches of sophorolipid samples were analyzed, and the similarity values of all fingerprints were found to be 0.965 or higher. Little differences in chemical composition were observed among the different batches of sophorolipid samples, and the quality of the sophorolipids was relatively consistent. The fingerprint established in this study is stable and reliable; it can be used for the quality evaluation of sophorolipids and lays a solid foundation for future research on production technology and the development and utilization of sophorolipids. The successful application of a universal CAD to the fingerprint establishment of sophorolipids also provides a reliable solution for the fingerprint establishment of substances with no or weak ultraviolet absorption.

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Oligomers are a particular category of non-intentionally added substances (NIAS) that may be present in food contact materials (FCMs), such as polyethylene terephthalate (PET), and consequently migrate into foods. Here, an ultra-high-pressure liquid chromatography quadruple time-of-flight mass spectrometry (UHPLC-qTOF-MS) method was developed for the analysis of 1st series cyclic PET oligomers in virgin olive oil (VOO) following a QuEChERS clean-up protocol. Oligomer migration was evaluated with two different migration experiments using bottles from virgin and recycled PET: one with VOO samples stored in household conditions for a year and one using the food simulant D2 (95% v/v ethanol in water) at 60 °C for 10 days. Calibration curves were constructed with fortified VOO samples, with the LOQs ranging from 10 to 50 µg L-1 and the recoveries ranging from 86.6 to 113.0%. Results showed no migration of PET oligomers in VOO. However, in the simulated study, significant amounts of all oligomers were detected, with the migration of cyclic PET trimers from recycled bottles being the most abundant. Additional substances were tentatively identified as linear derivatives of PET oligomers. Again, open trimer structures in recycled bottles gave the most significant signals.

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Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of specific regulating actions of enzymes, such as tyrosine kinases phosphorylating tyrosine residues or by nonenzymatic reactions, such as oxidation related to oxidative stress and diseases. While many studies have addressed the multisite, dynamic, and network-like properties of PTMs, only little is known of the interplay of the same site modifications. In this work, we studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which tyrosine residues were replaced with l-DOPA. The phosphorylated peptides were identified by liquid chromatography-high-resolution mass spectrometry and the site of phosphorylation by tandem mass spectrometry. The results clearly show that the oxidized tyrosine residues are phosphorylated, displaying a specific immonium ion peak in the MS2 spectra. Furthermore, we detected this modification in our reanalysis (MassIVE ID: MSV000090106) of published bottom-up phosphoproteomics data. The modification, where both oxidation and phosphorylation take place at the same amino acid, has not yet been published in PTM databases. Our data indicate that there can be multiple PTMs that do not exclude each other at the same modification site.

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Gandou decoction (GDD) is a traditional Chinese medicine prescription that has been widely used to treat copper metabolism disorders in China with remarkable clinical effect and lower toxicity. However, evaluation of the complexation ability of copper ions is challenging, which hinders screening and discovery of coordinate active ingredients in GDD. An analytical method is needed to determinate the complexation ability of chemical constituents with copper ions. In this study, a rapid and accurate method based on ultra-high performance liquid chromatography (UHPLC) was developed to determine the complexing ability of rhubarb with copper ions. First, the optimal coordination reaction conditions between active ingredients of rhubarb and copper ions were determined. The samples were separated using an Agilent Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 µm) with 5 µL injection volumes. The mobile phase was gradient eluted with methanol and water containing 0.1% (v/v) phosphoric acid at a flow rate of 0.3 mL/min. The detection wavelength was 254 nm and the column temperature was 30 ℃. Under the optimized chromatographic conditions, the rhubarb constituents were effectively separated. Next, peak areas of rhubarb were calculated before and after the coordination reaction between copper ions. The complexing ability of active ingredients in rhubarb with copper ions was evaluated by calculating the rate of changes of their chromatographic peak areas. Finally, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to identify the coordination active ingredients in rhubarb extract. Focusing on the coordination reaction conditions between active ingredients of rhubarb and copper ions revealed that the active ingredients of rhubarb and copper ions reached equilibrium by coordination reaction at pH 9 for 12 h. Methodological evaluation revealed the good stability and repeatability of the method. Under these conditions, 20 major components of rhubarb were identified by UPLC-Q-TOF-MS. According to the coordination rate of each component and copper ions, eight components with strong coordination were screened out (gallic acid 3-O-ß-D-(6'-O-galloyl)-glucopyranoside, aloe emodin-8-O-ß-D-glucoside, sennoside B, l-O-galloyl-2-O-cinnamoyl-glucoside, chysophanol-8-O-ß-D-(6″-O-acetyl)-glucoside, aloe-emodin, rhein and emodin). The respective complexation rates of the components were 62.50%, 29.94%, 70.58%, 32.77%, 34.61%, 26.07%, 28.73% and 31.78%. Compared with other reported methods, the presently developed method can be used to screen the active ingredients of traditional Chinese medicines that have complexing ability with copper ions, especially in complex mixture systems. This study describes an effective detection technology for evaluating and screening the complexing ability of other traditional Chinese medicines with metal ions.

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Plant components from extracts of Sophora flavescens, rhodiola, ginseng, Centella asiatica, and tea play important roles in skin whitening, moisturizing, anti-aging, sun protection, anti-inflammation, antiseptic, bacteriostatic, and other effects of cosmetics. At present, no relevant standard methods have been established to detect the addition amounts of plant extracts in cosmetics. In addition, plant extracts listed in product labels may be undetectable due to their addition in trace quantities and the lack of technical support. Therefore, a quantitative method for the simultaneous determination of 22 functional components in cosmetics was established by ultra-high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ/Orbitrap MS). Target compounds were extracted with methanol from samples using ultrasonic extraction, and then separated on a C18 column (100 mm × 2.1 mm, 1.8 µm) with gradient elution of 0.1% (v/v) formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program were as follows: 0-5 min, 5%B-8%B; 5-25 min, 8%B-60%B; 25-35 min, 60%B-80%B; 35-36 min, 80%B-5%B; 36-45 min, 5%B. The flow rate was 0.3 mL/min and the injection volume was 5 µL. Accurate masses of precursor ions were used to detect cosmetic functional components in positive ionization mode. The fragment ions obtained by higher energy collisional dissociation were used for confirmation of the functional components. Each compound showed good linearity. The limits of detection (LODs) were in the range of 0.003-2.01 mg/kg, and the limits of quantification (LOQs) were in the range of 0.02-4.36 mg/kg. Recoveries at three levels were 63.2%-125.1%, and relative standard deviations (RSDs) were 0.18%-10.9%. Fifty-four batches of samples labeled with four monomer functional components and nine plant extracts were tested. In the 17 batches of samples labeled with nicotinamide, 4 batches labeled with caffeine, and 6 batches labeled with Sophora flavescens root extract, the labeled functional components were detected. One out of 11 batches of samples labeled with D-panthenol was not detected. Three of the seven batches of samples labeled with ascorbyl glucoside were not detected. In the 21 batches of samples labeled with licorice extracts, the corresponding functional components were not detected in 9 batches. In the 21 batches of samples labeled with Centella asiatica extract, the corresponding functional components were not detected in 11 batches. In the 13 batches of samples labeled with tea extract, the corresponding functional components were not detected in 8 batches. In 11 of the 12 batches containing ginseng root extract, the corresponding functional components were not detected. In five of the six batches of astragalus membranaceus root extract samples, the corresponding functional components were not detected. In samples labeled with Polygonum cuspidatum root extract, Rehmannia glutinosa root extract, and Ophiopogon japonicus root extract, the corresponding functional components were detected. The method is simple, rapid, reliable, accurate, and suitable for the determination of the 22 functional components in cosmetics.

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The dark brown mixture resulting from the autooxidation of catechinic acid (CA) (AOCA) has been reported to possess antiviral activity against Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2). Unfortunately, the constituents of AOCA were not separated or identified and the compound(s) responsible for AOCA's antiviral activity remained unknown until recently. Colorless 4-hydroxy benzoic acid (4-HBA) has been reported as the main constituent (75%) of AOCA, and as being responsible for its antiviral activity. The findings seemed not to be reliable because of the existence in the literature of very different findings, because of the high concentration that was attributed to the supposed 4-HBA in the dark mixture, and because of the absence of essential analytical experiments to confirm 4-HBA in AOCA. Particularly, the AOCA chromatograms highlighting a peak attributable to 4-HBA, using commercial 4-HBA as a standard, is missing, as well as investigations concerning the antiviral activity of marketed 4-HBA. Therefore, in this study, to verify the exactness of the recent reports, we prepared CA from catechin and AOCA from CA, and the absence of 4-HBA in the mixture was first established by thin-layer chromatography (TLC), and then was confirmed by UHPLC­MS/MS, UV­Vis, and ATR­FTIR analyses. For further confirmation, the ATR­FTIR spectral data were processed by principal components analysis (PCA), which unequivocally established strong structural differences between 4-HBA and AOCA. Finally, while the antiviral effects of AOCA against HSV-2 were confirmed, a commercial sample of 4-HBA was completely inactive.

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A novel method based on ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) was developed for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods. In addition, the key points of the database establishment and application are summarized. This research focused on the derivatives of illegally added drugs. An HRMS database was established by comparing the response intensity of each compound in the positive and negative modes. The experimental conditions such as the type of extraction solvent and chromatographic column temperature were explored in detail. The analytes were separated on a Hypersil gold vanquish column (100 mm×2.1 mm, 1.9 µm) by gradient elution with acetonitrile/water (containing 0.1%(v/v) formic acid) as the mobile phase at a flow rate of 0.3 mL/min. Positive and negative ion full scanning/data-dependent secondary scanning mode was used to collect the 32 target compounds within 17 min, and TraceFinder software was used to screen the fragment ions. All the 32 compounds could be well separated within 17 min. The measured and theoretical values of the exact mass of the 32 compounds in the two matrix-spiked solutions were within an error of 5×10-6, and the MS2 fragment ions were within an error of 1×10-5. All the compounds showed an excellent linear relationship, with correlation coefficients (r2) above 0.99. Except dapoxetine, hydroxythiohomo sildenafil, thiohomo sildenafil, thiosildenafil, desmethyl thiosildenafil, the recoveries ranged from 50.5% to 84.5% in the solid matrix, with the relative standard deviations (RSDs) ranging from 1.2% to 13%. The recoveries were 60.4% to 109.3% in the liquid matrix, with the RSDs ranging from 0.77% to 8.2%. The matrix effect (ME) values of the 32 compounds ranged from 0.61 to 0.95 in the solid matrix and from 0.73 to 1.09 in the liquid matrix. Thiohomo sildenafil, desmethyl thiosildenafil, and chlorpretadalafil exhibited strong matrix inhibitory effects in the solid matrix. Therefore, solid and liquid negative matrix extracts were used to prepare a series of mixed standard solutions in order to reduce the ME values. The limits of detection (LODs) were 0.02 mg/kg for the 32 drugs in the liquid sample and 0.02 mg/kg for 29 compounds in the solid sample; the LODs for chlorothalidone, udenafil, and desmethyl thiosildenafil in the solid sample were 0.04 mg/kg. When the retention time in the self-built database matches the sample collection method, it should be used as one of the screening conditions. As for the selection of the matching mode, if the identify mode is selected, the retention time is a necessary condition for compound confirmation. When the retention time does not meet the requirements, subsequent screening of the fragment ions and isotope abundance ratios will not be performed. If the confirm mode is selected, the retention time is the optional condition for compound confirmation. When the retention time does not meet these requirements, subsequent matching of other conditions such as fragment ions and isotope information is required. Isotope information is very important in HRMS and is an effective supplement to the first-order extracted mass. Therefore, its use is recommended, but the isotope abundance ratio will be even lower when the target content is very low in the complex matrix, which may affect isotope matching. In addition, if the fragment ions are not detected in the screening results of the TraceFinder software but can be extracted in the data browser, their intensity threshold in the screening conditions can be further reduced to find the corresponding fragment ions. One positive sample was detected among 48 healthy food samples, with a detection rate of 2.08%. This method has the advantages of simple operation and high accuracy. It can be used for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods.

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Anthocyanins are the primarily pigments in many flowers, vegetables, and fruits and play a critical role in human and plant health. They are polyphenolic pigments that are soluble in water and usually quantified by spectrophotometric methods. The two main methods that quantify anthocyanins are pH differential and organic solvent-based methods. Our hypothesis was that these methods extract different anthocyanin profiles. Therefore, this experiment was designed to identify anthocyanin profiles that are extracted by pH differential and organic solvent-based methods and observe their total anthocyanin content from strawberries. Six methods were tested in this experiment to quantify and profile anthocyanins in strawberry fruits by spectrophotometry and Ultra High Performance Liquid Chromatography (UHPLC) respectively. Four methods used organic solvents (methanol, and chloroform-methanol) in different combinations. The next two methods were pH differential and a combination of organic solvent and the pH differential method. The results suggest that acidified chloroform-methanol extracted the highest anthocyanin content compared to water-based solvents. Methanol-water based solvents also performed better than methanol alone, because both methanol and water may extract different profiles of anthocyanins. Water-based extracts had the greatest absorbance at a lower wavelength (498 nm), followed by methanol (508 nm), and chloroform (530 nm). Chloroform-methanol solvent with higher pH (3.0) extracted pelargonidin as the main anthocyanin, while methanol and water-based solvents (with lower pH 1.0-2.0) extracted delphinidin as their main anthocyanin as identified by UHPLC. Therefore, chloroform-methanol and methanol-water solvents were the best solvents for extracting anthocyanins from strawberries. Also, freeze-dried strawberries had higher anthocyanin contents compared to fresh or frozen samples.

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An accurate mass database and a method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) were developed. These were applied in the screening and identification of illegally added medicines in herbal tea. Based on investigations, 167 medicines were selected to build an accurate MS database; these medicines included antipyretic analgesics, glucocorticoids, antibiotics, and antihistamines, among other categories. The database was established using Orbitrap HRMS and TraceFinder software. The database carried information on all selected compounds, including the molecular formula, accurate mass of precursor ions and fragment ions, retention time, and mass spectra. The samples were ultrasonically extracted with a 50% (v/v) methanol aqueous solution. The extracted solutions were separated using a Waters XBrigde BEH C18 column (100 mm×2.1 mm, 2.5 µm). As the mobile phases, 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid were used, with gradient elution. The sample solutions were analyzed by Orbitrap HRMS in the full-scan MS and data-dependent MS/MS acquisition modes (Full MS/dd-MS2). Positive and negative polarity data were simultaneously acquired. Some parameters were optimized to increase the peak intensity and sensitivity of all compounds. The resolutions in the full-MS scan and dd-MS2 scan were set to 70000 and 17500, respectively. In the full-MS mode, scanning was performed in the range of m/z 100 to 1000. In the MS/MS mode, the normalized collision energy (NEC) was set to 20%, 40%, and 60% for each compound. The inclusion list was not used during the measurement, and the dynamic exclusion time was set to 10.0 s. The loop count was set to 5. After acquiring the sample data with these conditions using Orbitrap MS, they were imported into TraceFinder software, through which the sample information was extracted and automatically matched with the information on compounds in the MS database. Screening and identification were conducted by comparing the retention times as well as the exact masses of precursor ions and fragment ions that were experimentally measured. If the errors between the experimentally and theoretically obtained masses of the precursor ions were below 5×10-6 and the deviations in retention times were less than 20 s, then suspicious positive compounds might be identified. Furthermore, if such compounds possess more than one similar fragment ion with a mass tolerance below 5×10-6, and exhibit similar ion distributions in the MS/MS profiles (compared to those in the database), they could be confirmed to be the same. The validation result showed that all compounds had good linear relationships, with correlation coefficients (r) greater than 0.99. Because pefloxacin, norfloxacin, desloratadine, astemizole and clindamycin had background interference, the method was not suitable for their quantification. Following experiments using three spiked concentrations, the recoveries of the rest 162 compounds were found to be in the range of 66.4%-118.1%, and the relative standard deviations (RSDs, n=6), in the range of 0.1%-16.1%. When the limit of detection (LOD) was 0.2 mg/kg, 83 compounds were detected, while when the LOD was 1.0 mg/kg, 167 compounds were detected. All compounds were matched successfully to the standard added sample with the MS database in TraceFinder software. To lower the likelihood of false positive and false negative results, a quality control method was recommended. The method was applied to analyze 245 herbal tea samples, among which 12 positive samples were detected. Thirteen positive compounds were found, including acetaminophen, diclofenac sodium, chlorpheniramine, brompheniramine, dexamethasone, dexamethasone 21-acetate, prednisone, prednisone 21-acetate, metronidazole, erythromycin, ciprofloxacin, amantadine, and dextromethorphan. In particular, amantadine, dextromethorphan, brompheniramine, and ciprofloxacin were newly detected, compared to standard methods. The developed method is rapid and accurate, and will be useful in the high-throughput screening of illegally added medicines in herbal tea.

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A method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) was established for the determination of genotoxic impurities 2, 6, and 12 in nifedipine. After extraction with methanol, the sample was injected into the UHPLC-Orbitrap HRMS system for analysis. An ACE EXCELTM 3 C18-AR column (150 mm×4.6 mm, 3 µm) was used for chromatographic separation. The mobile phase was methanol-0.1% formic acid aqueous solution (65∶35, v/v). The flow rate was 0.6 mL/min, while the column temperature and autosampler temperature were set as 35 ℃ and 8 ℃, respectively. The divert valve switching technique was used to protect the mass spectrometer. The six-way valve was set to divert the eluent of 7.5-11.6 min to waste and the rest of the eluent into the mass spectrometer. The Orbitrap mass spectrometer was coupled with the UHPLC system by an electrospray ion (ESI) source. The sheath gas and auxiliary gas flow rates were 60 and 20 arb (arbitrary units), respectively. The spray voltage was 3.5 kV, while the capillary temperature and auxiliary gas heater temperature were set as 350 ℃ and 400 ℃, respectively. The positive ion parallel reaction monitoring (PRM) scanning mode was adopted, and the mass spectral resolution was set to 35000 FWHM. The accurate masses of the [M+H]+ precursor ions of impurities 2, 6, and 12 were m/z 347.1230, 361.1026, and 347.1230, respectively. The accurate masses of the extracted [M+H]+ fragment ions of impurities 2, 6, and 12 were m/z 315.0968, 298.1069, and 315.0968, respectively. The normalized collision energies (NCEs) were optimized to 10%, 42%, and 10% for impurities 2, 6, and 12, respectively. The external standard method was utilized for quantitative analysis. The established method was validated in detail by investigating the specificity, linear range, limit of detection (LOD), limit of quantification (LOQ), recovery, precision, and stability. This method had good specificity, and the solvent did not interfere with the determination of impurities. The peak areas of impurities 2, 6, and 12 as well as their concentrations showed good linear relationships in the ranges of 0.2-100 ng/mL, with all correlation coefficients (r)≥0.9998. The recoveries of impurities 2, 6, and 12 at three levels (low, medium, and high) were in the range of 96.9%-105.0%, while the RSDs were between 1.21% and 5.12%. The LODs were 0.05 ng/mL and the LOQs were 0.2 ng/mL for all three impurities. This analytical method was used to determine impurities 2, 6, and 12 in three batches of nifedipine samples. Impurity 6 was not detected in the three batches, but impurities 2 and 12 were detected in all the three samples, and the detection amount was within the limit. The developed method is sensitive, fast, accurate, and easy to operate. It can provide a reference for the quality control of nifedipine by pharmaceutical companies and extend strong technical support for the supervision by drug regulatory authorities.

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Food contact materials (FCM) polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) used extensively in food packaging may contain cyclic oligomers which may migrate into food and thus cause toxic effects on human health. A simple, fast, and sensitive ultra-high-performance liquid chromatography method quadrupole time-of-flight mass spectrometer was developed for the analysis of 7 cyclic oligomers in post-mortem blood samples. The targeted analytes were separated on a Waters BEH C18 (150 × 2.1 mm, 1.7 µm) analytical column by gradient elution. Calibration curves were constructed based on standard solutions and blood samples and Student's t-test was applied to evaluate the matrix effect. The LODs ranged from 1.7 to 16.7 µg mL-1, while the method accuracy was assessed by recovery experiments and resulting within the range 84.2-114.6%. Such an analytical method for the determination of PET and PBT cyclic oligomers in biological samples is reported for the first time. The developed methodology allows the determination of these oligomers in blood providing a useful analytical tool to assess the exposure and thus the potential hazard and health risks associated with these non-intentionally added substances (NIAS) from PET and PBT FCM through food consumption. The method was validated and successfully applied to the analysis of 34 post-mortem whole blood samples. Polyethylene terephthalate trimer was detected in four of them, for the first time in literature.

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Fat-Soluble Vitamers [FSV] deficiencies and hypervitaminosis are associated with lifestyle diseases such as cardiovascular disease, diabetes, and cancer. Quantification of FSV and their metabolites in plasma has proved to be one of the most demanding analytical chemistry challenges. Current FSV quantification methods are compromises between breadth of coverage and sensitivity across the physiological range. Here, we developed and validated a sensitive, robust, semi-automated method using liquid-liquid extraction coupled with LC-ESI-MS/MS to quantify 11 FSV across their physiological concentrations in plasma. The addition of Phree® phospholipid removal plates as the last step in the extraction process reduced matrix effects, improving precision, recoveries, and the method's final sensitivity. This method can detect and quantify: retinol, retinoic acid, retinyl palmitate, 25 hydroxyvitamin D3 [25-OH-D3], 1-α-25-dihydroxy-D3, α-tocopherol, γ-tocopherol, α-tocotrienol, phylloquinone [K1], Menatetrenone [MK-4], and menaquinone-7 [MK-7].The Instrument Quantitation Limit [IQL]s for retinol (64.1 ng/mL), 25-OH-D3 (10.2 ng/mL), and α-tocopherol (3000 ng/mL) can detect clinical deficiencies. Our automated method will assist in the understanding of the complex interaction between these compounds and their possible role in health and disease.

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Cannabidiol (CBD), cannabinol (CBN), and Δ9-tetrahydrocannabinol (THC) are the most important components of hemp, whose concentrations determine the properties and applications of hemp. Hemp contains a large number of impurities, which must be removed from the extracting solution before determining the cannabinol contents by ultra-high performance liquid chromatography (UHPLC). Neutral alumina, magnesium silicate, and graphitized carbon black have different surface characteristics when used as adsorbents. The removal rates of pigments, total sugar, total fatty acid glyceride, and metal ions as well as the recoveries of the three cannabinols in the extraction solution were evaluated. The amounts of neutral alumina, magnesium silicate, and graphitized carbon black were 1.80 g, 0.15 g, and 0.05 g, respectively. The three adsorbents were mixed well and packed into a polypropylene pipe to prepare a special 2 g/6 mL solid phase extraction (SPE) column for determining the three cannabinol compounds in hemp. The chemical components of the hemp flowers and leaves were extracted with an ethyl acetate/methanol (9∶1, v/v) mixture. After the extracting solution was allowed to pass through the SPE column, the recoveries of CBD, CBN, and Δ9-THC were 98.9%, 95.7%, and 99.2%, respectively. The removal rates of xanthophyll, chlorophyll a, and chlorophyll were 96.3%, 99.2%, and 95.5%, respectively. The removal rates of total sugar, total fatty glyceride, and metal ions were 98.5%, 96.9%, and 85.4%, respectively. In this study, the chromatographic conditions for analyzing the three cannabinol compounds were optimized. The cannabinol compounds were separated within 10 min on an Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 µm) using a mobile phase consisting of 1% (v/v) acetic acid and acetonitrile (30∶70, v/v) at a flow rate of 0.5 mL/min. The detection wavelength was set at 210 nm with a diode array detector, and the sample injection volume was 1 µL. Good linear relationships were observed between the mass peak areas and mass concentrations of CBD, CBN, and Δ9-THC in the range of 0.5-50 mg/L. The corresponding correlation coefficients (R2) were 0.9983, 0.9995, and 0.9981, while the detection limits were 0.45 µg/L, 0.53 µg/L, and 0.38 µg/L. The recoveries of CBD, CBN, and Δ9-THC were 90.3%-96.9%, 93.7%-95.6%, and 90.8%-96.1%, with relative standard deviations (RSDs) of 2.2%-6.1%, 4.1%-8.0%, and 2.4%-4.8%, respectively. The results were satisfactory, demonstrating that the special SPE column made of neutral alumina, magnesium silicate, and graphitized carbon black was well suited for the determination of the three cannabinol compounds in hemp.

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A fast and simple ultra-high-performance liquid chromatography using diode array detector (UHPLC-DAD) technique has been developed for the simultaneous determination of 22 sunscreen agents (UV filters), viz. phenylbenzimidazole sulfonic acid (PBS), terephthalydene dicamphor sulfonic acid (TDS), benzophenone-4 (BZ4), camphor benzalkonium methosulfate (CBM), benzophenone-2 (BZ2), benzylidene camphor sulfonic acid (BCS), benzophenone-3 (BZ3), 3-benzylidene camphor (3BC), isoamyl p-methoxycinnamate (IMC), 4-methylbenzylidene camphor (MBC), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), octocrylene (OCR), butyl methoxydibenzoyl methane (BDM), ethylhexyl dimethyl PABA (EDP), ethylhexyl methoxycinnamate (EMC), homosalate (HS), ethylhexyl salicylate (ES), diethylhexyl butamido triazone (DBT), ethylhexyl triazone (ET), drometrizole trisiloxane (DRT), methylene bis-benzotriazolyl tetramethylbutylphenol (MBP), and bis-ethylhexyloxyphenol methoxyphenyl triazine (EMT) in cosmetic products. Different parameters, such as column type, oven temperature, mobile phase composition, and detection wavelength, were studied. The best chromatographic separation was obtained under the following conditions: Poroshell 120 EC-C18 (100 mm×4.6 mm, 2.7 µm) column set at 25 ℃ and gradient acetonitrile-isopropanol-water (containing 50 mmol/L ammonium acetate and 0.05% (v/v) formic acid) as the mobile phase, pumped at a flow rate of 0.5 mL/min, with a wavelength of 311 nm. The proposed UHPLC-DAD technique provided separation of the 22 target sunscreen agents within 35 min, with the optimized sample pretreatment procedure below. First, samples were mixed thoroughly by adding 2 mL or 5 mL tetrahydrofuran, followed by vortex and dispersal. If the wax samples were still not homogenized completely, an ultrasonic dispersal protocol with heating to 50 ℃ was adopted. Second, the homogeneous samples were ultrasonically extracted with ethanol containing 0.1% (v/v) formic acid. The developed method showed good linear relationships, with correlation coefficients of no less than 0.998. Two kinds of samples with different matrix types were fortified at three levels. The average spiked recoveries of 22 UV filters ranged from 85.2% to 112.3%, with the relative standard deviations (RSDs) ranging from 0.5% to 6.5%. The limits of detection were between 0.7 and 64 mg/kg, and the limits of quantification ranged from 2.4 mg/kg to 215 mg/kg. Moreover, the stabilities of the mixed standard solutions at the levels of 2, 10, and 50 mg/L were tested. The stability results showed that drometrizole trisiloxane was stable for 12 h, while the others were stable for 36 h. The reliability of the developed method was demonstrated by applying it to 16 commercial sunscreen-containing cosmetic samples obtained from the Chinese market. The levels determined in this study agreed well with those of five commercial samples (such as emulsion and cream). The method developed was remarkably different from the standard method, which is mentioned in the Safety and Technical Standards for Cosmetics (2015 edition), especially in terms of mobile phase composition and extraction solvent. Compared to the standard method, this method bypassed the use of large amounts of corrosive solvents like tetrahydrofuran and perchloric acid, thus improving the extraction efficiency of low-polarity components like drometrizole trisiloxane, methylene bis-benzotriazolyl tetramethylbutylphenol, and bis-ethylhexyloxyphenol methoxyphenyl triazine, and the analytes were well separated with better stability. Benzophenone-2 was added to this method as another detection component. The good analytical features, as well as their environment-friendly characteristics, make the presented method suitable not only for routine analysis in cosmetics industries, but also as a candidate reference method for sunscreen analysis.

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BACKGROUND: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. RESULTS: We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). CONCLUSIONS: Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.

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In this work, pipette-tip micro-solid phase extraction (PT-µSPE) which packed by melamine-foam@polydopamine (MF@PDA) coupled with ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF) was developed for extraction and determination of psychotropic drugs in serum samples. Considering the operation back pressure, the melamine-foam as carrier material with 3D cross-linked grid structure can provide high permeability and contact surface. MF@PDA was prepared by self-polymerization reaction of dopamine under weak alkaline conditions and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). The surface group of PDA containing catechol structure, quinone structure and amine group has multi-interaction with psychotropic drugs which can increase the adsorption capacity. Moreover, the parameters affecting extraction efficiencies such as extraction and desorption cycle, pH value, eluent type, ionic strength and amount of sorbent were investigated. Based on the high sensitivity and accuracy mass measurement by TOF/MS, under the optimized extraction condition, the limits of detection (LOD) of this method were obtained in the range of 0.002-0.1 ng ml-1. The linearity was ranged from 0.01 ng ml-1 to 600 ng ml-1, and all the correlation coefficients (R2) were above 0.993. The spiked recoveries were in the range of 80.04% to 109.18% in real sample test and RSD values obtained from 0.95% to 9.85%. The results demonstrate that MF@PDA-PT-µSPE-UHPLC-QTOF is a sample and reliable method for the detection of psychotropic drugs in serum sample.

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An Ultra High-Performance Liquid chromatography method quadruple time-of-flight mass spectrometry has been developed for the analysis of 11 cyclic polyesters oligomers, following a modified QuEChERS clean-up with alumina/primary secondary amine, in pasta. Target analytes were polyethylene terephthalate (PET) 1st series cyclic dimer to heptamer, polybutylene terephthalate (PBT) dimer to pentamer and a polyurethane oligomer. Standard addition method was applied for the calibration, and the limits of quantification ranged from 3.2 to 17.2 ng g-1. Recoveries ranged from 86.4 to 109.8%, RSDs were lower than 12% for all analytes, and matrix effect never exceeded ± 2.5%. The method was successfully applied to real commercial pasta samples, where the PET 1st series cyclic trimer was the most abundant oligomer, being found in all tested samples. The 1st series PET cyclic dimer and tetramer, as well as 1,4,7-trioxacyclotridecane-8,13-dione, were found in considerable amounts. Traces of the 2nd and 3rd series PET cyclic dimers were also found.

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