bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒07‒11
twenty-nine papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 25. pii: S1570-0232(21)00327-5. [Epub ahead of print]1179 122846
      Simultaneous determination of kynurenines, neurotransmitters, pterins and steroids linked to various neurological and metabolic diseases have important diagnostic significance for related pathology and drug monitoring. An improved, sensitive and selective ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometric (UHPLC-MS/MS) method, based on our earlier publication, has been proposed for the quantitative measurement of 42 metabolites in human urine. The assay covers a larger number of analytes, uses an advanced, Waters Atlantis T3 chromatographic column and similarly meets the guideline of European Medicines Agency (EMA) on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all > 0.998. Intra-day and inter-day accuracy and precision were 87-118%, 81-120% and 2-20%, respectively including the lower limit of quantification (LLOQ). The assay is expected to facilitate the diagnosis and allows drug level monitoring from urine.
    Keywords:  Kynurenine; LC-MS/MS; Neurotransmitter; Pterin; Urine
  2. Se Pu. 2021 Jul 08. 39(7): 695-701
      Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 μm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 μm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R2) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.
    Keywords:  high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); muscle relaxants; pancuronium; quaternary ammonium; rocuronium; vecuronium
  3. Food Funct. 2021 Jul 06.
      Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 μM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.
  4. Se Pu. 2021 Jan;39(1): 69-76
      Measurement of organophosphate esters (OPEs) in human body fluids is important for understanding human internal exposure to OPEs and for assessing related health risks. Most of the current studies have focused on the determination of OPE metabolites in human urine, as OPEs are readily metabolized into their diester or hydroxylated forms in the human body. However, given the existence of one metabolite across multiple OPEs or multiple metabolites of one OPE, as well as the low metabolic rates of several OPEs in in vitro studies, the reliability of urinary OPE metabolites as biomarkers for specific OPEs is needs to be treated with caution.Human blood is a matrix that is in contact with all body organs and tissues, and the blood levels of compounds may better represent the doses that reach target tissues. Currently, only a few studies have investigated the occurrence of OPEs in human blood by different analytical methods, and the variety of OPEs considered is limited. In this study, a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of 16 OPEs in human blood, and the extraction efficiency of the solid phase extraction (SPE) column for OPEs was verified. To human blood samples, 10 ng of an internal standard was added, followed by mixing and aging for 30 min. The samples were extracted three times with acetonitrile using a shaker, and then purified on ENVI-18 cartridges with acetonitrile containing 25% dichloromethane as the eluent. Finally, the OPEs were analyzed by high performance liquid chromatography-tandem mass spectrometry. After optimization of the analytical column and mobile phases, the analytes were separated on a BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution using methanol and 5 mmol/L ammonium acetate in water as the mobile phase. Then, the analytes were ionized in electrospray ionization positive (ESI+) mode and detected in the multiple reaction monitoring (MRM) mode. The mass spectral parameters, including the precursor ion, product ion, declustering potential, entrance potential, and collision cell exit potential, were optimized. The results were quantified by the internal standard method. The limits of detection (LOD, S/N=3) of the OPEs were in the range of 0.0038-0.882 ng/mL. The calibration curves for the 16 OPEs showed good linear relationships in the range of 0.1-50 ng/mL, and the correlation coefficients were >0.995. The extraction efficiency of the ENVI-18 column for the 16 OPEs was validated, and the average recoveries of the target compounds were 54.6%-104%. The average recoveries (n=3) of 15 OPEs, except trimethyl phosphate (TMP), in whole blood at three spiked levels were in the range of 53.1%-126%, and the relative standard deviations (RSDs) were in the range of 0.15%-12.6%. The average recoveries of six internal standards were in the range of 66.8%-91.6% except for TMP-d9 (39.1%), with RSDs of 3.52%-6.85%. The average matrix effects of the OPEs in whole blood were 56.4%-103.0%. Significant matrix effects were found for resorcinol bis(diphenyl phosphate) (RDP) (75.8%±1.4%), trimethylphenyl phosphate (TMPP) (68.4%±1.0%), 2-ethylhexyl di-phenyl phosphate (EHDPP) (56.4%±12.4%), and bisphenol-A bis(diphenyl phosphate) (BABP) (58.5%±0.4%). However, these effects could be corrected by similar signal suppressions of the corresponding internal standard (TPHP-d15, 77.4%±7.5%). This method is simple, highly sensitive, and suitable for the determination of OPEs in human blood. Fifteen human whole blood samples were collected to quantify the 16 OPEs using the developed method. The total concentrations of the OPEs ranged from 1.50 to 7.99 ng/mL. The detection frequencies of eight OPEs were higher than 50%. Tri-iso-butyl phosphate (TiBP), tri(2-chloroethyl) phosphate (TCEP), and tri(1-chloro-2-propyl) phosphate (TCIPP) were the dominant OPEs, with median concentrations of 0.813, 0.764, and 0.690 ng/mL, respectively. These results indicated widespread human exposure to OPEs, which should be of concern.
    Keywords:  high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); human blood; liquid-liquid extraction (LLE); organophosphate ester; solid phase extraction (SPE)
  5. Nat Methods. 2021 Jul;18(7): 779-787
      Chimeric MS/MS spectra contain fragments from multiple precursor ions and therefore hinder compound identification in metabolomics. Historically, deconvolution of these chimeric spectra has been challenging and relied on specific experimental methods that introduce variation in the ratios of precursor ions between multiple tandem mass spectrometry (MS/MS) scans. DecoID provides a complementary, method-independent approach where database spectra are computationally mixed to match an experimentally acquired spectrum by using LASSO regression. We validated that DecoID increases the number of identified metabolites in MS/MS datasets from both data-independent and data-dependent acquisition without increasing the false discovery rate. We applied DecoID to publicly available data from the MetaboLights repository and to data from human plasma, where DecoID increased the number of identified metabolites from data-dependent acquisition data by over 30% compared to direct spectral matching. DecoID is compatible with any user-defined MS/MS database and provides automated searching for some of the largest MS/MS databases currently available.
  6. Methods Mol Biol. 2021 ;2314 549-577
      Decades of study have highlighted the richness and uniqueness of the repertoire of lipid and glycolipid families produced by mycobacteria. Many of these families potently regulate host immune responses, in stimulatory or suppressive ways. Thus, the global study of this repertoire in different genetic backgrounds or under model conditions of infection is gaining interest. Despite the difficulties associated with the specificities of this repertoire, the field of mass spectrometry-based lipidomics of mycobacteria has recently made considerable progress, particularly at the analytical level. There is still considerable scope for further progress, especially with regard to the development of an efficient bioinfomatics pipeline for the analysis of the large datasets generated. This chapter describes an HPLC-MS methodology allowing the simultaneous screening of more than 20 of the lipid families produced by mycobacteria and provides recommendations to analyze the generated data given the state-of-the-art.
    Keywords:  HPLC-MS; Lipidomics; Mass spectrometry; Mycobacterium
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jun 03. pii: S1570-0232(21)00302-0. [Epub ahead of print]1178 122821
      Anti-doping substances listed by the World Anti-Doping Agency (WADA) include hundreds of compounds of very different physico-chemical properties. Anti-doping control laboratories need to screen all these substances in the so-called Initial Testing Procedures (ITPs) what is very challenging from an analytical point of view. ITPs are mostly based on reversed-phase (RP) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using C18 columns, which feature poor retention and peak tailing for polar and basic compounds, respectively. While studies on this field dealing with the comparison of stationary phases are focused on certain chemical classes, this research provides a wide multi-target approach. For this purpose, a representative group of 93 anti-doping agents (log P from -2.4 to 9.2) included in ten different classes of prohibited substances was selected. A comprehensive study on the performance of six columns and four eluents on different separation parameters (retention factors, asymmetry factors, co-elutions, total run times) and matrix effects (signal enhancement or suppression) was performed for LC-MS/MS-based ITPs. Columns working in both RP [C18, C8, phenyl hexyl (PH), pentafluorophenyl (PFP) and mixed-mode hydrophilic/RP (HILIC-RP)) and hydrophilic (HILIC)] modes were investigated. Eluents contained methanol or acetonitrile as organic modifiers, with or without the addition of ammonium acetate. The best column-mobile phase binomial for ITPs was PFP using water-methanol (0.1% formic acid) as eluent, while HILIC was the best option for highly polar non-aromatic anti-doping agents, which were poorly addressed by PFP. Excellent good peak shapes and relative acceptable matrix interferences were obtained for HILIC-RP, which was tested for the first time for the analysis of anti-doping agents, although the number of compounds eluting too fast was too high. On the whole, the alkyl phase C18 showed the worst performance and although C8 and PH were better, their performance did not surpass that of PFP. Possible retention mechanisms underlying separation in the different stationary phases were discussed. This research provides valuable information to anti-doping control labs for improving LC-MS/MS-based ITPs and it proposes PFP as a suitable alternative to the already established C18.
    Keywords:  Anti-doping control; Column comparison; Initial testing procedures; Liquid chromatography; Mass spectrometry
  8. Se Pu. 2021 May;39(5): 518-525
      Tryptophan (Trp), also known as α-amino β-indolepropionic acid, is an essential amino acid, which is involved in various physiological processes. Studies have shown that tumors, infectious diseases, and neurological diseases are accompanied by Trp-related metabolic disorders. Understanding the excretion of Trp and its metabolites in normal individuals is of great significance for treating Trp-related diseases and monitoring the health. A rapid quantitative method was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Further, this method was applied to the simultaneous determination of Trp and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA). The excretion and amount of target compounds in random urine samples collected from healthy participants were studied using this method. Urine samples were collected from healthy male volunteers (between 20-22 years old) without any diet and exercise restrictions. Urine samples were collected between 11∶00-13∶00 daily for 10 d. Thereafter, the urine samples were diluted, centrifuged, and subjected to pre-column derivatization with dansyl chloride (DNS-Cl). Caffeic acid (CA) was used as the internal control. Later, the derivatives were detected using triple quadrupole mass spectrometry with electron pray ionization (ESI) in positive and multi reaction monitoring (MRM) modes. The samples were separated using a Thermo C18 column (50 mm×3 mm, 2.7 μm) with 0.1% aqueous formic acid aqueous solution and methanol as mobile phases at a flow rate of 0.2 mL/min. The three most abundant ions for each derivative were selected for downstream analysis, and the internal control was used for quantification. The polarity and molecular weight of the compounds were found to be altered effectively after DNS-Cl derivatization treatment. The dansyl group effectively altered the polarities of the derivatives, such that their retention behaviors in the reverse elution system were similar and they were well separated. The interference due to impurities was effectively eliminated using the MRM mode. The results showed significant linear correlation, since the correlation coefficients were greater than 0.9740. The recoveries were between 93.24%-107.65%, and the LODs were 0.005-0.5 ng/mL for the eight compounds. Trp prototype and the seven target metabolites, including 3-OH-Kyn, 3-OH-AA, XA, Kyn, KA, 5-HIAA, and 5-HT generated through Trp-5-HT and Trp-Kyn pathways were detected in the urine samples. These results indicated that Trp was excreted in a prototypic form or after being metabolized. The level of the target compounds in random urine samples of individuals were 0.99-3.72 (3-OH-Kyn), 2.51-21.11 (3-OH-AA), 0.25-1.12 (XA), 0.15-1.53 (Kyn), 0.24-2.58 (KA), 0-0.31 (5-HT), and 2.2-17.94 (5-HIAA) μg/mL. For the same individual, in the state of physical health, the fluctuations of Trp and its metabolites in urine were large. Due to these large fluctuations in the absolute content, the difference between individuals was not significant. The data generated using 70 urine samples revealed that the amount of excreted Trp being metabolized was 124%-268% of prototype, which further indicated that the excretion after metabolism was the major underlying mechanism. Upon comparing the levels of metabolites in the Trp-5-HT and Trp-Kyn pathways, the results indicated that the levels of 3-OH-AA and 3-OH-Kyn generated upon Trp degradation through the Kyn pathway was higher than those of the other products. Trp was degraded via Kyn pathway to produce 3-OH-AA, which was the main metabolite of Trp found to be present in the body. This manuscript detected the levels of Trp and its metabolites, as well as summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.
    Keywords:  pre-column derivation; tryptophan-kynurenine pathway; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); urine
  9. Eur Rev Med Pharmacol Sci. 2021 Jun;pii: 26149. [Epub ahead of print]25(12): 4394-4399
      OBJECTIVE: New fentanyl analogues have been constantly emerging into the illegal drug market as cheap substitutes of heroin posing a serious health threat for consumers because of their high toxicity. Analytical methods to disclose the presence of these compounds in biological fluids of intoxicated individuals need to be updated to keep up with the new trends. In this study, we updated an ultra-high-performance liquid chromatography-tandem mass spectrometry method previously developed, for detecting some new fentanyl analogues and metabolites (sufentanil and norsufentanil, cis-3-methylnorfentanyl, trans-3-methylnorfentanyl, metabolites of cis and transmethylfentanyl, beta-phenylfentanyl, phenylfentanyl, para-fluoro furanyl fentanyl, isobutyryl fentanyl and ocfentanil) in urine sample.MATERIALS AND METHODS: Urine samples were simply diluted before injection in the chromatograph equipped with a reversed phase microcolumn. Detection was achieved with a triple quadrupole mass spectrometer with an electrospray ionization source in positive ion mode and operated in multiple reaction monitoring.
    RESULTS: The chromatographic separation was short (5 min) and the method was fully validated with a high sensitivity being limits of quantifications from 0.003 to 0.006 µg/L urine for the analytes under investigation.
    CONCLUSIONS: The suitability of the method was tested with urine specimens from former heroin addicts, which resulted positive by immunological screening to the class of fentanyl analogues. This method represents a valid tool to document recent exposure to the above-reported compounds for clinical and forensic purposes.
  10. Se Pu. 2021 Apr 08. 39(4): 391-398
      Urine is an important source of biomolecular information for metabolomic studies. However, the acquisition of high-quality metabolomic datasets or reliable biomarkers from urine is difficult owing to the large variations in the concentrations of endogenous metabolites in the biofluid, which are caused by diverse factors such as water consumption, drugs, and diseases. Thus, normalization or calibration is essential in urine metabolomics for eliminating such deviations. The urine osmolality (Π), which is a direct measure of the total urinary solute concentration and is not affected by circadian rhythms, diet, gender, and age, is often considered the gold standard for estimation of the urine concentration. In this study, a pre-data acquisition calibration strategy based on osmolality was investigated for its feasibility to overcome sample concentration variability. Before data acquisition, the product of the osmolality×injection volume of all samples was set to be equivalent through the uses of a customized injection volume or dilution. After ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) analysis of the sample, the raw dataset was normalized to the total ion abundance or total useful MS signals (MSTUS) to achieve further calibration. The osmolality of each urine sample was determined with a freezing-point depression osmometer. For the instrumental analysis, a Vanquish UPLC system coupled to a Q-Exactive Plus HRMS device was used for metabolite analysis and accurate mass measurement. Full-scan mass spectra were acquired in the range of m/z 60-900, and the MS/MS experiments were conducted in "Top5" data-dependent mode. A Waters UPLC column (100 mm×2.1 mm, 1.8 μm) was used for chromatography separation. The raw data were imported into Progenesis QI software for peak picking, alignment, deconvolution, and normalization. SIMCA-P software was used for the principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA). This strategy was first applied to sequentially diluted urine samples, where three frequently used normalization methods were compared. In the identical injection volume experiment, the points were scattered and showed relevant distribution according to the dilution multiple in the plot of PCA scores. There was little improvement after normalization to either the total ion abundance or MSTUS. In the customized injection volume experiment, the urine samples derived from the same source showed ideal clustering. With total ion abundance and MSTUS normalization, the dataset was further improved in the PCA model fitting and prediction. As a result, there were more peaks with a peak area RSD of <30%, which indicated better parallelism. The diluted urine solutions had higher Spearman's coefficient values with their sample source than those without calibration, which suggested less intra-group differences. The strategy was further validated using data from a metabolomic study of children with congenital hydronephrosis and healthy controls. As a concentration estimator, osmolality showed better linear correlation with the mass signal and was less influenced by physiological or pathological factors, thus obtaining broader application and more accurate results than creatinine. The concentration variability was effectively eliminated after customized dilution calibration and showed a more obvious clustering effect in the PCA score plot. The OPLS-DA-based statistical model used to identify discriminate metabolites was improved, with less chance of overfitting. In conclusion, the calibration strategy based on osmolality combined with total ion abundance or MSTUS normalization significantly overcame the problem of urine concentration variability, eliminated intra-group differences, and possessed better parallelism, thus giving better clustering effects in PCA or OPLS-DA and higher reliability of the statistical model. The results of this study provide guidance and a reference for future metabolomic studies on urine.
    Keywords:  metabolomics; normalization; osmolality; ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS); urine
  11. Anal Chem. 2021 Jul 06.
      Epoxy/dihydroxy-oxylipins are important biologically active compounds that are mainly formed from polyunsaturated fatty acids (PUFAs) in the reactions catalyzed by the cytochrome P450 (CYP 450) enzyme. The analysis of epoxy/dihydroxy-oxylipins would be helpful to gain insights into their landscape in living organisms and provide a reference for the biological studies of these compounds. In this work, we employed chemical labeling-assisted liquid chromatography (LC) coupled with high-resolution mass spectrometry (CL-LC-HRMS) to establish a highly sensitive and specific method for screening and annotating epoxy/dihydroxy-oxylipins in biological samples. The isotope reagents 2-dimethylaminoethylamine (DMED) and DMED-d4 were employed to label epoxy/dihydroxy-oxylipins containing carboxyl groups so as to improve the analysis selectivity and MS detection sensitivity of epoxy/dihydroxy-oxylipins. Based on a pair of diagnostic ions with a mass-to-charge ratio (m/z) difference of 15.995 originating from the fragmentation of derivatives via high-energy collision dissociation (HCD), the potential epoxy/dihydroxy-oxylipins were rapidly screened from the complex matrix. Furthermore, the epoxy/dihydroxy groups could be readily localized by the diagnostic ion pairs, which enabled us to accurately annotate the epoxy/dihydroxy-oxylipins detected in biological samples. The applicability of our method was demonstrated by profiling epoxy/dihydroxy-oxylipins in human serum and heart samples from mice with high-fat diet (HFD). By the proposed method, a total of 32 and 62 potential epoxy/dihydroxy-oxylipins including 42 unreported ones were detected from human serum and the mice heart sample, respectively. Moreover, the relative quantitative results showed that most of the potential epoxy/dihydroxy-oxylipins, especially the oxidation products of linoleic acid (LA) or α-linolenic acid (ALA), were significantly decreased in the heart of mice with HFD. Our developed method is of high specificity and sensitivity and thus is a promising tool for the identification of novel epoxy/dihydroxy-oxylipins in biological samples.
  12. Se Pu. 2021 Apr 08. 39(4): 406-414
      Chloramphenicols, nitroimidazoles, lincosamides, and macrolides are common antibiotics used in veterinary medicine. Overdoses of these drugs will lead to residual substances in animal-derived foods and accumulate in the body through the food chain, thereby exerting adverse effects on human health. Therefore, regulation of veterinary drug levels is imperative to ensure the quality of animal-derived foods and safeguard the health of consumers. In this study, a method based on ultra performance liquid chromatography-tandem mass spectrometry coupled with solid phase extraction (SPE-UPLC-MS/MS) was developed for the simultaneous determination of eight prohibited and restricted veterinary drugs and three metabolite residues across four categories (chloramphenicols, nitroimidazoles, lincosamides, and macrolides) in eggs, liquid milk, chicken, and freshwater fish. The main factors affecting the response, recovery, and sensitivity of the method, such as the type and pH values of the extraction solvent, dilution solution for the analytes, type of chromatographic column, and type and proportion of the mobile phase, were optimized during sample pretreatment and instrument analysis. The samples were hydrolyzed and dispersed in 0.1 mol/L phosphate buffer solutions (pH 9.0) and extracted with acetonitrile. The extract was further extracted using ethyl acetate. After centrifugation, the supernatant ethyl acetate was concentrated to near dryness in nitrogen below 40 ℃. The residue was dissolved in 0.3 mL methanol, followed by the addition of 5.7 mL phosphate buffer solution. After shaking, the solutions were purified and enriched on an Oasis HLB SPE column. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm) at a column temperature of 40 ℃ with a flow rate of 0.4 mL/min. The injection volume was 10 μL. Gradient elution was carried out with methanol and 0.1% formic acid aqueous solution as the mobile phases. Multiple reaction monitoring (MRM) was conducted in the positive and negative electrospray ionization modes. The isotope internal standard method was used for quantitative analysis. Under optimal conditions, each analyte showed a good linear relationship in each range, and the correlation coefficient (R2) was greater than 0.99. The limits of detection (LODs) ranged from 0.050 to 0.50 μg/kg, and the limits of quantification (LOQs) ranged from 0.20 to 1.5 μg/kg. With eggs, freshwater fish, chicken, and liquid milk as the matrix samples, the recoveries in spiked blank samples were determined at different addition levels in compliance with the current legislation. The average recoveries of the 11 analytes were 65.3% to 108%. The relative standard deviations (RSDs) were between 0.40% and 21%. The matrix effects of the analytes were between 0.0124% and 46.80% in four different samples after purification on the Oasis HLB column. The practicality of the proposed approach for routine analyses of the eight prohibited and restricted veterinary drugs, and three metabolite residuals was evaluated by applying it to the determination of these compounds in animal-derived food samples. The samples, including 80 eggs, 80 chicken, 40 liquid milk, and 32 freshwater fish, were procured from a supermarket and a farm product market. The results of the positive samples were consistent with those observed with the standard methods. The method described herein is easy to operate, sensitive, and accurate. It is suitable for the simultaneous and rapid determination of various prohibited and restricted veterinary drug residues and metabolites in animal-derived foods.
    Keywords:  animal-derived foods; isotope internal standard; matrix effect (ME); metabolites; solid phase extraction (SPE); ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS); veterinary drug residues
  13. J Mass Spectrom. 2021 May 27. 56(8): e4772
      Advances in high-resolution, nontargeted mass spectrometry allow for the simultaneous measure of thousands of metabolites in a single biosample. Application of these analytical approaches to population-scale human studies has been limited by the need for resource-intensive blood sample collection, preparation, and storage. Dried blood spotting, a technique developed decades ago for newborn screening, may offer a simple approach to overcome barriers in human blood acquisition and storage. In this study, we find that over 4,400 spectral features across diverse chemical classes may be efficiently and reproducibly extracted and relatively quantified from human dried blood spots using nontargeted metabolomic analysis employing HILIC and reversed-phase liquid chromatography coupled to Orbitrap mass spectrometry. Moreover, over 80% of metabolites were found to be chemically stable in dried blood spots stored at room temperature for up to a week. In direct relation to plasma samples, dried blood spots exhibited comparable representation of the human circulating metabolome, capturing both known and previously uncharacterized metabolites. Dried blood spot approaches provide an opportunity for rapid and facile human biosampling and storage and will enable widespread metabolomics study of populations, particularly in resource-limited areas.
    Keywords:  chromatography; dried blood spots; metabolomic analysis; nontargeted mass spectrometry; sample preparation
  14. Se Pu. 2021 Jul 08. 39(7): 744-749
      An analytical method was established for the simultaneously determination the pentostatin and 2'-amino-2'-deoxyadenosine contents in fermentation broth by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After high-speed centrifugation, aqueous solution dilution, vortex shock, and microfiltration, the fermentation broth samples were analyzed by HPLC-MS/MS. The samples were separated on a Waters Atlantis® T3 column (100 mm×2.1 mm, 5 μm) using a gradient elution program with 10 mmol/L ammonium formate (containing 0.1% formic acid) and methanol (containing 0.02% formic acid) as the mobile phases. Moreover, a chromatographic protection column (5 mm×2.1 mm, 5 μm) was added to preserve the column efficiency. The flow rate, column temperature, and injection volume were set at 0.3 mL/min, 25 ℃, and 10 μL, respectively. Qualitative and quantitative analyses of the target compounds were performed using an ESI+ source. MS parameters such as the collision energies and tube lens offsets of pentostatin and 2'-amino-2'-deoxyadenosine were optimized. The quantitative ion pairs of pentostatin and 2'-amino-2'-deoxyadenosine were m/z 269.17>153.20 and m/z 267.00>136.10, respectively; the corresponding collision energies were 11 V and 18 V. The external standard method was used for quantitative analysis. The established method was verified rigorously in terms of the linear range, limit of detection, limit of quantification, recovery rate, and precision. Pentostatin and 2'-amino-2'-deoxyadenosine showed good linear relationships in the range of 1.0-250 μg/L. The correlation coefficients ranged from 0.9969 to 0.9996, and the relative standard deviations (RSDs) ranged from 6.51% to 8.35% (n=8). This result indicated good accuracy and exactitude in the detection of the pentostatin and 2'-amino-2'-deoxyadenosine. The recoveries (n=6) at three spiked levels (1.0, 5.0, and 25 μg/L) were in the ranges of 97.94%-104.46% and 89.96%-107.21% for the pentostatin and 2'-amino-2'-deoxyadenosine, respectively; the corresponding RSDs were in the ranges of 3.74%-4.88% and 4.81%-13.29%. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) of the 2'-amino-2'-deoxyadenosine and pentostatin in the fermentation broth were 0.003-0.060 μg/L and 0.010-0.200 μg/L, respectively. The validated experimental method was used for the detection of actual samples, viz. the stored multiple pentostatin-producing mutagenic strains in our laboratory. The HPLC-MS/MS method for the determination of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth offered the advantages of small sampling volume, strong maneuverability, good stability, and high sensitivity. Compared with previously published methods, this systematically established and optimized method significantly reduced the detection time, and matrix effects were well suppressed. Moreover, the peak shape and stability of the target compounds were greatly improved. This method provides a methodological basis and meaningful reference for the detection of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth.
    Keywords:  2'-amino-2'-deoxyadenosine; fermentation broth; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); pentostatin
  15. Ther Drug Monit. 2021 Jun 28.
      BACKGROUND: Recent studies indicate that a high proportion of patients in the intensive care unit (ICU) fail to attain adequate antibiotic levels. Thus, there is a need to monitor the antibiotic concentration to ensure effective treatment. Herein, the authors aimed to develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of antimicrobials to assess individualized therapeutic drug monitoring (TDM).METHODS: A UHPLC-MS/MS method with 11 antibiotics (ciprofloxacin, moxifloxacin, benzylpenicillin, levofloxacin, linezolid, rifampicin, meropenem, cloxacillin, cefotaxime, clindamycin, and piperacillin) was developed. Chromatographic separation was performed using a Kinetex biphenyl reversed-phase column, with gradient elution using 0.1% formic acid (FA) and methanol with 0.1% FA. Sample preparation was performed using methanol protein precipitation. The total run time was 5 min.
    RESULTS: For all analytes, the inter-assay inaccuracies for calibrators were ≤5%. The inter-day inaccuracies for the quality controls (QCs) were ≤5% for all analytes. The inter-assay precision for calibration standards ranged between 1.42% and 6.11%. The inter-assay imprecision for QCs of all antibiotics and concentrations ranged between 3.60% and 16.1%. Inter-assay inaccuracy and imprecision for the QCs and calibration standards were ≤15% for all drugs, except benzylpenicillin.
    CONCLUSION: A rapid UHPLC-MS/MS method was developed for the simultaneous quantification of 11 different antibiotics. Minimal sample preparation was required to ensure a rapid turnaround time. The method was applied to clinical samples collected from four ICUs.
  16. Se Pu. 2021 May;39(5): 552-557
      Sunscreens can be categorized as physical and chemical types. Chemical sunscreens are widely used in cosmetics, and hence, their concentration in the desired products should be strictly monitored. Gas chromatography-mass spectrometry (GC-MS) is widely used for the analysis of cosmetics as it does not require organic mobile phases and allows for accurate qualitative and quantitative analyses. In this study, a method based on GC-MS was established for the determination of 13 sunscreen agents in cosmetics: ethylhexyl salicylate, homosalate, 4-methylbenzylidene camphor, ethylhexyl dimethyl para-aminobenzoic acid, ethylhexyl methoxycinnamate, octocrylene, butyl methoxydibenzoylmethane, diethylamino hydroxybenzoyl hexyl benzoate, 3-benzylidene camphor, benzophenone-3, camphor benzalkonium methosulfate, drometrizole trisiloxane, and isopentyl-4-methoxycinnamate. Accordingly, 0.5 g of the cosmetic product was dissolved in dichloromethane in a 50 mL volumetric flask and extracted ultrasonically for 15 min. Then, 1.0 mL of the extracting solution was withdrawn and diluted to 50.0 mL with dichloromethane. The mixture was filtered through a 0.22-μm membrane. A 1 μL aliquot of the dichloromethane solution was introduced onto the HP-5ms chromatographic column (30 m×250 μm×0.25 μm). The 13 components were separated under programed temperature elevation in the interval from 150 ℃ to 290 ℃. These components could be analyzed within 30 min after being ionized by the EI source, and their determination was achieved in selected ion monitoring (SIM) mode. The external standard method was employed for quantitative determination. Linear equations, linear correlation coefficients, and linear ranges were obtained by analyzing a series of mixed standard working solutions. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the 13 sunscreen agents were determined. The matrix effect and average recoveries of the 13 agents with six extraction solvents (dichloromethane, tetrahydrofuran, methanol, acetonitrile, n-hexane, and acetone) were compared. Among these, dichloromethane showed a weak matrix effect and high average recovery. The matrix effect of dichloromethane was 90.1%-100.5%, and the average recovery was 96.3%. All the 13 sunscreen agents showed good linearity in their corresponding ranges. The correlation coefficients (r 2) were higher than 0.998. The LODs and LOQs were in the ranges of 0.04 to 0.63 mg/g and 0.12 to 2.10 mg/g, respectively. Two types of cosmetics were selected to verify the accuracy and precision of the method at three levels. The average spiked recoveries of the 13 sunscreen agents in cream and lotion were 88.7%-103.6%, and 88.4%-102.3%, respectively; the corresponding relative standard deviations (RSDs, n=6) were 1.7%-4.9% and 1.2%-3.9%. Whitening cosmetics are frequently added with sunscreen agents, which is a regulatory blind spot. Five batches of skin whitening products containing sunscreen agents were detected using this method. The contents of five sunscreen agents in skin whitening cosmetics ranged from 0.8% to 5.2%, which were lower than the relevant limits in China. Owing to its advantages of simple operation, high sensitivity, and good recovery, the proposed method is suitable for the qualitative and quantitative determination of 13 sunscreen agents in cosmetics. This method provides technical support for market supervision and laboratory testing.
    Keywords:  cosmetics; gas chromatography-mass spectrometry (GC-MS); sunscreen agents
  17. Se Pu. 2021 May;39(5): 488-493
      Measurement of additive residues in food contact materials is important for safety monitoring at the initial stage. Most of the current studies focus on the determination of the migration amounts of chemical hazards from food contact materials into food simulants. Studies on chemical hazard residues in food contact materials are limited to monomers, oligomers, heavy metals, phthalic acid esters, and biphenols, which are known environmental pollutants. Only a few studies have investigated analysis methods for additive residues in food contact materials. In this study, the main factors (monitoring wavelength, chromatographic column, mobile phase, extraction solvent, etc.) that affect the accuracy and sensitivity of eight compounds, including three antioxidants, three light stabilizers, and two plasticizers, were investigated during sample preparation and instrument analysis. A method based on ultrahigh-performance liquid chromatography (UPLC) was developed for the simultaneous determination of these eight additives in polyethylene (PE). The PE food contact material sample was ground to homogenize the particle sizes under freeze-grinding. After comparing the extraction efficiencies of methylbenzene, chloroform, acetone, and acetonitrile, 2.0 g of the sample was extracted with methylbenzene at 80 ℃ and 10.34-11.72 MPa (1500-1700 psi) by accelerated solvent extraction (ASE) for 10 min once. The exaction solvent (10 mL) was transferred and concentrated to near dryness under a gentle stream of nitrogen gas and then re-dissolved in 10 mL of the initial mobile phase (70% (v/v) methanol in water). Finally, the eight compounds were analyzed by UPLC. After optimization of the analytical column and mobile phases, the eight analytes were separated on an ACQUITY UPLC BEH C8 chromatographic column (100 mm×2.1 mm, 1.7 μm) by gradient elution using water and acetonitrile as the mobile phases. The column oven temperature, flow rate of the mobile phase, and injection volume were 30 ℃, 0.3 mL/min, and 5 μL, respectively. The analytes were detected by a diode assay detector (DAD) in the scanning range of 210 nm to 400 nm. The monitoring wavelength was set at 230 nm, 250 nm, 280 nm, and 330 nm. External standard calibration curves were used for quantification. Under the optimized conditions, the calibration curves for the eight compounds showed good linearity in the range of 0.2 μg/mL to 10 μg/mL, and the correlation coefficients were >0.999. The recoveries in spiked blank polyethylene samples at the level of 0.05% were in the range of 83.8% to 103.4%, with relative standard deviations (RSDs) ranging from 0.14% to 7.86%. To validate the method, PE reference materials containing these eight compounds were manufactured at the content level of 0.2% to 0.9%. The recoveries using the prepared reference materials ranged from 63.5% to 118.5%, and the RSDs were in the range of 4.61% to 15.6%. The limits of detection (LODs, S/N=3) of all the eight compounds were 0.005% and the limits of quantification (LOQs, S/N=10) were 0.02%, in compliance with the current legislation. To assess the feasibility and potential of the proposed approach for routine analyses of these eight compounds, the developed method was applied to the analysis of these compounds in ten PE food packages and PE gloves. In six samples, tris(2,4-di-tert-butylphenyl)phosphite (Irganox 168) was detected at a level of 0.02%-0.07%, which was lower than the maximum level of this compound in PE food contact material products regulated in GB 9685-2016 at 0.2%. The method is compliant with the current legislation, and it can be used for the monitoring and supervision of these eight additives in PE food contact materials.
    Keywords:  additive; food contact material; polyethylene (PE); ultrahigh-performance liquid chromatography (UPLC)
  18. Se Pu. 2021 May;39(5): 472-477
      In recent years, goat milk powder and camel milk powder have gained popularity among consumers. Due to their potential low allergenicity, these milk powders have become a substitute for breast milk, especially for infants, and for people with lactose intolerance. In this paper, a method was developed for the simultaneous determination of 18 amino acids (AAs), histidine (His), serine (Ser), arginine (Arg), glycine (Gly), aspartic acid (Asp) combined with asparagine (Asn), glutamic (Glu), glutamine (Gln), threonine (Thr), alanine (Ala), proline (Pro), lysine (Lys), tyrosine (Tyr), methionine (Met), valine (Val), isoleucine (Iso), leucine (Leu), and dimer of cysteine (Cys) combined with cysteine (L-Cys-Cys), phenylalanine (Phe), taurine (Tau) in milk, goat milk, and camel milk power. The aim of the research was to compare the three kinds of milk powder from the perspective of the constituent amino acids. Therefore, the amino acid compositions and contents were compared. Thus, 2.0 g of the sample was accurately weighed, added to 16 mL H2O, and mixed thoroughly. Then, 200 mg of the sample was weighed in a glass tube with a stream of nitrogen to displace oxygen. The samples were hydrolyzed in HCl for 24 h at 110 ℃. Then, the amino acids were pre-column derivatized by 6-aminoquinoline-n-hydroxysuccinimide carbamate (AQC). In precolumn derivatization combined with reverse-phase chromatography, both 2,4-dinitrofluorobenzene (DNFB) and phenylisothiocyanate (PITC) can react with primary amines and secondary amines. However, the derivatization time is approximately 1 h. In contrast, the derivatization time of AQC was greatly shortened. Derivatization led to the conversion of free amino acids into highly stable derivatives, which were separated by ultra performance liquid chromatography (UPLC) with UV detection at 260 nm and quantified by the external standard method. The samples were separated on a BEH C18 column (150 mm×2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min. The calibration curves showed good linearity, with correlation coefficients greater than 0.999. The limits of detection (LODs) and limits of quantification (LOQs) of the 18 amino acids were 1.3-2.5 (mg/100 g) and 3.9-7.5 (mg/100 g), respectively. Quality control samples of SRM 1849a were used as the reference material. The results were in accordance with the content range. The RSDs ranged from 2.04% to 3.65%. Furthermore, the developed method was successfully applied to determine the types and concentrations of amino acids in 11 samples purchased from local markets in Shanghai and online shops. Abundant amino acids were detected in the three types of milk powder. While all the milk powder samples contained 18 types of amino acids, Tau was not detected in some of the goat and camel milk powder samples. Total essential amino acids (TEAA) in total amino acids (TAA) of milk powder was the highest of all. The TEAA values of TAA in the goat and camel milk powders were similar. The developed method requires only 22 min for the separation of 18 amino acids. This method is suitable for the large-scale analysis of milk powder samples, and it demonstrates high sensitivity and accuracy for the determination and confirmation of the 18 amino acids in different types of milk powders.
    Keywords:  6-aminoquinoline-n-hydroxysuccinimide carbamate (AQC); amino acids; camel milk powder; goat milk powder; milk powder; pre-column derivatization; ultra performance liquid chromatography (UPLC)
  19. J Diabetes Metab Disord. 2021 Jun;20(1): 591-599
      Background: Amino acids (AAs) and acylcarnitines play a key role in metabolic disease and can be used as biomarkers of various diseases such as malignancies, type 2 diabetes (T2D), insulin resistance, and cardiovascular diseases, therefore, designing an accurate and simple laboratory method that simultaneously measure both groups of substances, could improve the process of analytes quantification. In this research, a flow injection tandem mass spectrometry (FI-MS/MS) method for simultaneous measurement of AAs and acylcarnitines in addition to results of validation is explained.Methods: Samples were mixed with internal standards and after derivatization (with butanolic-HCL), AAs, and acylcarnitines were quantified by tandem mass spectrometry (SCIEX API 3200). Analytical performance studies were designed based on the Clinical and Laboratory Standards Institute (CLSI) guidelines including precision, accuracy, linearity, and limit of detection-quantification (LOD-LOQ) experiments. Samples from patients with T2D in different stages of kidney disease were also analyzed to ensure the clinical usage of the method.
    Results: Performance evaluation of the method demonstrated adequate results. The mean of estimated inter-assay precision (reported as a coefficient variation) for AAs and acylcarnitines were less than 8.7% and 12.3%, the estimated mean bias was below 8.8% and 10.2% respectively. LOD of analytes ranged between 0.6-10 μmol per liter (μmol/L) for AAs and 0.02-1 μmol/L for acylcarnitines. LOQ analytes showed a range of 2-25 μmol/L and 0.05-5 μmol/L for AAs and carnitine/acylcarnitines respectively. In diabetic patients sample analysis, a significant increase in acylcarnitines (C2, C4, C5DC, C6, C8, C10, C14) and citrulline with a significant decrease in valine were seen in patients with severely increased albuminuria.
    Conclusion: FI-MS/MS method with pre-injection derivatization with butanolic-HCL can be used for concurrent measurement of AAs and carnitine/acylcarnitines in a short time and it satisfies the analytical performance requirements. This method is applied for AAs and carnitine/acylcarnitines measurement in patient with T2DM and results show some of the acylcarnitines and AAs can be involved in diabetic nephropathy development.
    Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-021-00786-3.
    Keywords:  Acylcarnitine; Amino acid; Carnitine; Diabetes; Nephropathy; Tandem mass spectrometry
  20. Se Pu. 2021 Apr 08. 39(4): 399-405
      Lipophilic shellfish toxins pose significant threats to the health of seafood consumers and public health. The symptoms of these kinds of toxins include severe diarrhea, abdominal cramps, nausea and gastrointestinal disorders. These symptoms could be hardly distinguished with many other symptoms of food poisoning and diseases. Therefore, a fast and accurate determination method in human biological samples is urgently needed for the accurate judgement of food poisoning incident, which is important for the investigation of public health emergencies and clinical treatment of poisoned patients. However, there were several flaws of the previous studies reported on the analysis of lipophilic shellfish toxins: (1) limited target compounds were covered; (2) the pre-treatment process was complex; (3) the sensitivity of the compound was low. In this study, a simple extraction method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 12 lipophilic shellfish toxins, including azaspir acid 1 (AZA1), azaspir acid 2 (AZA2), azaspir acid 3 (AZA3), dinophysistoxin 1 (DTX1), dinophysistoxin 2 (DTX2), gymnodimine (GYM), hyessotoxin (HYTX), okadaic acid (OA), pinnatoxin (Pntx), pectenotoxins 2 (PTX2), spirolides 1 (SPX1), yessotoxin (YTX), in plasma and urine. Firstly, the instrument conditions were optimized. Different additions in mobile phase were compared and 0.05% (v/v) ammonia solution was selected since it can improve the peak shape of YTX and HYTX, and increase the respondence by four times. Secondly, the volume of acetonitrile (0.2, 0.4, 0.6, 0.8, 1.0 mL) use for the extraction of the target compounds in plasma was optimized. Satisfactory recoveries were obtained when 0.6 mL of acetonitrile was used. At the same time, satisfactory recoveries were obtained when 0.9 mL of acetonitrile was used in urine samples. Finally, under the optimized conditions, the 12 compounds in plasma and urine samples were ultrasonically extracted with acetonitrile. Chromatographic separation was performed on a Phenomenex Kinetex C18 column (50 mm×3 mm, 2.6 μm) with 90% (v/v) acetonitrile aqueous solution and water containing 0.05% (v/v) ammonia as mobile phases. Gradient elution with a flow rate of 0.40 mL/min was employed. The 12 compounds were monitored in the multiple reactions monitoring (MRM) mode with electrospray ionization (ESI) under both positive and negative conditions. The matrix effects of the 12 compounds ranged from 0.8 to 1.1. Therefore, external standard calibration curves were used for the quantification. The 12 shellfish toxins showed good linear relationship in the range of 0.03-36.25 μg/L with the correlation coefficients greater than 0.995. The limits of detection (LODs, S/N=3) were 0.08-0.21 ng/mL for the urine samples and 0.10-0.28 μg/L for the plasma samples, respectively. The limit of quantitations (LOQs, S/N=10) were 0.23-0.63 μg/L for the urine samples and 0.31-0.84 μg/L for the plasma samples, respectively. The recoveries of the 12 compounds were in the range of 72.7%-124.1% at three spiked levels (i. e., LOQ, three times LOQ, and ten times LOQ). The intra-day and inter-day precisions were 2.1%-20.0% and 2.1%-15.3%, respectively. The method was applied in the detection of the 12 lipophilic shellfish toxins in the urine and plasma samples of healthy humans and mice previously injected with the 12 shellfish toxins intraperitoneally. None of the 12 toxins were found in the samples from healthy human, while all of the 12 lipophilic shellfish toxins were found in the urine and plasma samples collected from the poisoned mice in the range of 1.14-2.35 μg/L and 1.01-1.17 μg/L, respectively. The established method has the advantages of sensitive, quick, easy to operate, and of low sample volume. It can be used for the simultaneous determination of 12 lipophilic shellfish toxins in urine and plasma samples.
    Keywords:  lipophilic shellfish toxins; plasma; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); urine
  21. Se Pu. 2021 Jan;39(1): 96-103
      In this study, a comprehensive analytical method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for the determination of nine N-nitrosamines in animal derived foods. There are many kinds of N-nitrosamines in foods that are harmful to human health. However, the national standard GB 5009.26-2016 pertains only to the detection of N-dimethylnitrosamine; there are many drawbacks of this method, such as complicated sample preparation, low recovery rate, and poor reproducibility. Hence, it is of practical significance to establish a method for the simultaneous determination of a variety of N-nitrosamines. The optimal extraction conditions for the developed method were as follows: 10.0 g aliquots of the sample were placed in a 50 mL centrifuge tube, followed by the addition of 10 mL acetonitrile and 200 μL internal working standard solutions. After 30 min of freezing treatment, 4 g magnesium sulfate and 1 g sodium chloride were added for dehydration, and the tube was centrifuged at 9000 r/min for 5 min. After vortex centrifugation, 5 mL of the clear supernatant was purified using 150 mg polystyrene divinylbenzene (PLS-A). The purified extracts were dewatered using 1.6 g MgSO4 and 0.4 g NaCl, and then filtered through a 0.22 μm membrane filter unit prior to GC-MS/MS analysis. Temperature-programmed was applied at an initial temperature of 50 ℃. After 0.16 min, the temperature was raised to 220 ℃ at the rate of 900 ℃/min for 5 min. N-Nitrosamines were separated on an HP-Innowax column (30 m×0.25 mm×0.25 μm). Identification and quantification were achieved using an electron impact ion (EI) source in positive ion mode with multiple reaction monitoring (MRM). The internal standard method was used to quantify the N-nitrosamines. Under the optimal conditions, the correlation coefficients of the standard calibration curves were not less than 0.99 in the range of 0.1-50.0 μg/L. The limits of detection were 0.03-0.30 μg/kg (S/N=3), and limits of quantification were 0.15-1.00 μg/kg (S/N=10). At spiked levels of 0.5, 1.0, and 3.0 μg/kg, the average recoveries of N-nitrosamines in spiked samples ranged from 80.4% to 98.5%, with relative standard deviations between 2.41% and 12.50%. This method was used to determine animal derived food products, except N-itrosomethylethylamine and N-nitrosomorpholine, others were founded. The results showed that N-nitrosamines levels in salted aquatic products were generally higher than those of the other samples. The method established in this study is simple to operate, and it does not require any time-consuming distillation extraction. Furthermore, there is minimal consumption of samples and reagents; consequently, the experiment cost is reduced, and the method is environmentally friendly. This method has theoretical and practical significance for the control of N-nitrosamines residues in animal derived foods, establishment of detection standards, and corresponding management measures.
    Keywords:  N-nitrosamines; QuEChERS; animal derived foods; gas chromatography-tandem mass spectrometry (GC-MS/MS)
  22. Se Pu. 2021 May;39(5): 526-533
      Ginsenosides are the main active compounds of ginseng, American ginseng and Panax notoginseng. They have certain pharmacological effects on the cardiovascular, immune and central nervous systems. Most ginsenosides are naturally classified as protopanaxatriol (PPT), protopanaxadiol (PPD), and oleanolic acid (OA) according to their aglycone skeletons. The nine main ginsenosides are Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2. Accurate quantification of ginsenosides is imperative because they are the characteristic components and quality evaluation indicators of health foods. A new method based on solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was developed for the determination of the nine ginsenosides in health foods. First, the pretreatment conditions were optimized. With the aim of purifying the samples and removing impurities, SPE cartridges with different packing materials, such as Alumina-N/XAD-2 SPE Cartridge, C18 and HLB were investigated. Based on the purification efficiencies, recoveries and other factors, the Alumina-N/XAD-2 SPE cartridge composite SPE column was selected as the pretreatment purification column. The eluents were then optimized. When water was used as the eluent, the ginsenosides could remain adsorbed on the SPE column, and could not be eluted down with other water-soluble substances. By increasing the proportion of ethanol in the eluent, the ginsenoside adsorbed on the filler of the SPE column could be gradually eluted. When the proportion of ethanol in the eluent reached 70%, the ginsenosides could be completely eluted. The effects of different volumes of 70% ethanol elution solvent (5-30 mL) on the extraction efficiencies of ginsenosides were also investigated. The results showed that when the volume of the elution solvent reached 20 mL, the ginsenosides were completely eluted. Then, the chromatographic conditions and MS parameters were optimized. By examining the ionization cracking of ginsenosides, the quasi-molecular ions and corresponding fragment ions in ginsenoside primary MS were determined. After optimizing the chromatographic conditions and MS parameters, not only the sensitivity of the method was improved, but also the isomers Rb2, Rb3 and Rc with the same quasi-molecular ions and the corresponding fragment ions were completely separated. Good separation was achieved for the nine ginsenosides, thus meeting the requirements for accurate quantification. Finally, chromatographic separation was achieved on a Hypersil Gold C18 column (100 mm×2.1 mm, 1.9 μm) under linear gradient elution using a 5 mmol/L ammonium acetate solution (with 0.1% formic acid) and acetonitrile as the mobile phases. The nine ginsenosides were detected using a triple quadrupole MS detector under ESI - and multiple reaction monitoring (MRM) modes, and quantified by the external standard method. The nine ginsenosides showed a strong positive linear correlation (r 2>0.9950) in the range of 0.005-0.5 μg/mL. The sample recoveries and the corresponding relative standard deviations (RSDs) were 81.1%-114.2% and 0.4%-8.0% (n=6), respectively. Eleven batches of health foods on the market, among which six batches contained ginseng, American ginseng or Panax notoginseng ingredients, were analyzed by the developed method, and the ginsenosides were detected. The total ginsenosides contents were close to those mentioned on the label. However, the nine ginsenosides were detected in one batch of health food, whose label did not indicated ginseng, American ginseng or Panax notoginseng. The nine ginsenosides were not detected in the remaining batches of health foods.The health food extract was directly loaded and purified without any complex pretreatment. The UPLC⁃MS/MS method, not only helped shorten the analysis time, but also accurate quantification of low ginsenoside contents in complex matrix samples. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the nine ginsenosides in health foods.
    Keywords:  ginsenosides; health foods; solid phase extraction (SPE); ultra performance liquid chromatography⁃tandem mass spectrometry (UPLC MS/MS)
  23. Prog Lipid Res. 2021 Jul 02. pii: S0163-7827(21)00030-8. [Epub ahead of print]83 101114
      Knowing the spatial location of the lipid species present in biological samples is of paramount importance for the elucidation of pathological and physiological processes. In this context, mass spectrometry imaging (MSI) has emerged as a powerful technology allowing the visualization of the spatial distributions of biomolecules, including lipids, in complex biological samples. Among the different ionization methods available, the emerging surface-assisted laser desorption/ionization (SALDI) MSI offers unique capabilities for the study of lipids. This review describes the specific advantages of SALDI-MSI for lipid analysis, including the ability to perform analyses in both ionization modes with the same nanosubstrate, the detection of lipids characterized by low ionization efficiency in MALDI-MS, and the possibilities of surface modification to improve the detection of lipids. The complementarity of SALDI and MALDI-MSI is also discussed. Finally, this review presents data processing strategies applied in SALDI-MSI of lipids, as well as examples of applications of SALDI-MSI in biomedical lipidomics.
    Keywords:  Biological tissues; Lipidomics; Lipids; Mass spectrometry imaging; SALDI
  24. Nat Methods. 2021 Jul;18(7): 799-805
      A growing appreciation of the importance of cellular metabolism and revelations concerning the extent of cell-cell heterogeneity demand metabolic characterization of individual cells. We present SpaceM, an open-source method for in situ single-cell metabolomics that detects >100 metabolites from >1,000 individual cells per hour, together with a fluorescence-based readout and retention of morpho-spatial features. We validated SpaceM by predicting the cell types of cocultured human epithelial cells and mouse fibroblasts. We used SpaceM to show that stimulating human hepatocytes with fatty acids leads to the emergence of two coexisting subpopulations outlined by distinct cellular metabolic states. Inducing inflammation with the cytokine interleukin-17A perturbs the balance of these states in a process dependent on NF-κB signaling. The metabolic state markers were reproduced in a murine model of nonalcoholic steatohepatitis. We anticipate SpaceM to be broadly applicable for investigations of diverse cellular models and to democratize single-cell metabolomics.
  25. J Proteome Res. 2021 Jul 06.
      Screening of characteristic biomarkers from chiral amino-containing metabolites in biological samples is difficult and important for the noninvasive diagnosis of gastric cancer (GC). Here, an enantiomeric pair of chlorine-labeled probes d-BPCl and l-BPCl was synthesized to selectively label d- and l-amino-containing metabolites in biological samples, respectively. Incorrect structural annotations were excluded according to the characteristic 3:1 abundance ratio of natural chlorine isotopes (35Cl and 37Cl) derived from the probes. A sensitive C18 HPLC-QQQ-MS/MS method in combination with the probes was then developed and applied in metabolomic analysis of amino-containing metabolites in urine samples. A total of 161 amino-containing metabolites were rapidly separated and determined, and 28 chiral amino acids and achiral glycine were quantified with good precision and accuracy. A total of 18 differential variables were discriminated by analyzing chiral amino-containing metabolites in urine samples of the GC patient and healthy person using the probe-based HPLC-MS/MS-MRM method combined with the orthogonal partial least squares discriminant analysis and Mann-Whitney U test with false discovery rate correction for multiple hypotheses. A diagnostic regression model including d-isoleucine, d-serine, and β-(pyrazol-1-yl)-l-alanine and age was then constructed with an average prediction correctness of 88.9% in the validation set. This work established a close connection between gastric cancer and chiral amino-containing metabolites. The mass spectrometry data analyzed in the study are publicly available via Mendeley Data (DOI: 10.17632/4bd93j9yrr.1).
    Keywords:  biomarkers; chiral amino-containing metabolites; chlorine-labeled probes; gastric cancer; metabolomics; urine
  26. Nat Methods. 2021 Jul;18(7): 747-756
      Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.
  27. Front Cell Dev Biol. 2021 ;9 682269
      The objective of this study was to identify potential biomarkers and possible metabolic pathways of malignant and benign thyroid nodules through lipidomics study. A total of 47 papillary thyroid carcinomas (PTC) and 33 control check (CK) were enrolled. Plasma samples were collected for UPLC-Q-TOF MS system detection, and then OPLS-DA model was used to identify differential metabolites. Based on classical statistical methods and machine learning, potential biomarkers were characterized and related metabolic pathways were identified. According to the metabolic spectrum, 13 metabolites were identified between PTC group and CK group, and a total of five metabolites were obtained after further screening. Its metabolic pathways were involved in glycerophospholipid metabolism, linoleic acid metabolism, alpha-linolenic acid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, Phosphatidylinositol signaling system and the metabolism of arachidonic acid metabolism. The metabolomics method based on PROTON nuclear magnetic resonance (NMR) had great potential for distinguishing normal subjects from PTC. GlcCer(d14:1/24:1), PE-NME (18:1/18:1), SM(d16:1/24:1), SM(d18:1/15:0), and SM(d18:1/16:1) can be used as potential serum markers for the diagnosis of PTC.
    Keywords:  lipidomics; orthogonal partial least square discriminant analysis; papillary thyroid carcinoma; pathway; plasma samples
  28. J Anal Methods Chem. 2021 ;2021 5570938
      Novel, accurate, selective, and rapid high-performance liquid chromatography mass spectrometry method was developed for simultaneous analysis of amoxicillin trihydrate, dicloxacillin sodium, and their official impurity 6-aminopenicillanic acid. The chromatographic separation was carried out by applying the mixture on a C18 column (3.5 µm ps, 100 mm × 4.6 mm id) using acetonitrile:water (65 : 35 by volume) as a mobile phase within only 4 min. The quantitative analysis was executed using single quadrupole mass spectrometer in which electrospray ionization, selected ion monitoring, and negative mode were operated. The retention times were 1.61, 2.54, and 3.50 mins for amoxicillin, 6-aminopenicillanic acid, and dicloxacillin, respectively. The method was validated in linear ranges of 2-28 µg mL-1, 2-35 µg mL-1, and 1-10 µg mL-1 for amoxicillin, dicloxacillin, and 6-aminopenicillanic acid, respectively. The results obtained from the suggested HPLC/MS were statistically compared with those obtained from the reported HPLC method, where no significant difference appeared respecting accuracy and precision. According to the analytical eco-scale assessment method, the proposed method was proved to be greener than the reported one, where the analysis time and the amount of the wasted effluent decreased.
  29. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Feb 04. pii: S1570-0232(21)00043-X. [Epub ahead of print]1178 122563
      Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC-MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4-1500 pmol/L for E1 and 4-2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC-MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC-MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.
    Keywords:  Estradiol; Estrogen; Estrone; Immunoassay; LC–MS/MS; Menopause; Reference values