bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒10‒18
fourteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Chromatogr A. 2020 Sep 28. pii: S0021-9673(20)30861-X. [Epub ahead of print]1632 461587
    Konya Y, Izumi Y, Bamba T.
      Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from -5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples.
    Keywords:  CO(2); Metabolomics; Polar compounds; Supercritical fluid chromatography; Tandem mass spectrometry; Trifluoroacetic acid
  2. Metabolites. 2020 Oct 08. pii: E398. [Epub ahead of print]10(10):
    B Gowda SG, Gowda D, Liang C, Li Y, Kawakami K, Fukiya S, Yokota A, Chiba H, Hui SP.
      Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C ≥ 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.
    Keywords:  Chemical labeling; DMED; FAHFAs; High-fat diet; LC-MS; murine model
  3. Molecules. 2020 Oct 12. pii: E4639. [Epub ahead of print]25(20):
    Klupczynska A, Misiura M, Miltyk W, Oscilowska I, Palka J, Kokot ZJ, Matysiak J.
      A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5-108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research.
    Keywords:  amino acids; cell culture; liquid chromatography-mass spectrometry; metabolomics; proline
  4. Metabolites. 2020 Oct 09. pii: E400. [Epub ahead of print]10(10):
    Aidaros AA, Sharma C, Langhans CD, G Okun J, Hoffmann GF, Dasouki M, Chakraborty P, Aljasmi F, Y Al-Dirbashi O.
      This article reports a targeted metabolomic method for total plasma fatty acids (FAs) of clinical or nutritional relevance. Thirty-six saturated, unsaturated, or branched-chain FAs with a chain length of C8-C28 were quantified using reversed-phase liquid chromatography-tandem mass spectrometry. FAs in plasma (10 μL) were acid-hydrolyzed, extracted, and derivatized with DAABD-AE (4-[2-(N,N-Dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole) at 60 °C for 1 h. Derivatization resulted in a staggering nine orders of magnitude higher sensitivity compared to underivatized analytes. FAs were measured by multiple-reaction monitoring using stable isotope internal standards. With physiological and pathological analyte levels in mind, linearity was established using spiked plasma. Intra-day (n = 15) and inter-day (n = 20) imprecisions expressed as variation coefficient were ≤10.2% with recovery ranging between 94.5-106.4%. Limits of detection and limit of quantitation ranged between 4.2-14.0 and 15.1-51.3 pmol per injection, respectively. Age-stratified reference intervals were established in four categories: <1 month, 1-12 month, 1-18 year, and >18 year. This method was assessed using samples from patients with disorders affecting FAs metabolism. For the first time, C28:0 and C28:0/C22:0 ratio were evaluated as novel disease biomarkers. This method can potentially be utilized in diagnosing patients with inborn errors of metabolism, chronic disease risk estimation, or nutritional applications.
    Keywords:  derivatization; inborn errors of metabolism; liquid chromatography-tandem mass spectrometry; plasma fatty acids; targeted metabolomics
  5. Anal Chem. 2020 Oct 13.
    Davis DE, Sherrod SD, Gant-Branum RL, Colby JM, McLean JA.
      Routine small-molecule analysis is challenging owing to the need for high selectivity and/or low limits of quantification. This work reports a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 14 antiepileptic drugs (AEDs) in human serum. For the optimized LC-MS/MS method described herein, we applied the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) LC-MS C62-A document and the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance for Industry to evaluate the quality of the assay. In these studies, AED linearity, analyte recovery, matrix effects, precision, and accuracy were assessed. Using liquid chromatography-drift tube ion mobility-mass spectrometry (LC-DTIM-MS), a qualitative method was also used to increase confidence in AED identification using accurate mass and collision cross section (CCS) measurements. The LC-DTIM-MS method was also used to assess the ability of drift tube CCS measurements to aid in the separation and identification of AED structural isomers and other AEDs. These data show that another dimension of information, namely CCS measurements, provides an orthogonal dimension of structural information needed for AED analysis. Multiplexed AED measurements using LC-MS/MS and LC-DTIM-MS have the potential to enable better optimization of dosing owing to the high precision capabilities available in these types of analytical studies. Taken together, these data also show the ability to increase confidence in small-molecule identification and quantification using these analytical technologies.
  6. Anal Chim Acta. 2020 Oct 16. pii: S0003-2670(20)30824-2. [Epub ahead of print]1134 125-135
    Neef SK, Winter S, Hofmann U, Mürdter TE, Schaeffeler E, Horn H, Buck A, Walch A, Hennenlotter J, Ott G, Fend F, Bedke J, Schwab M, Haag M.
      Formalin-fixed and paraffin-embedded (FFPE) tissue represents a valuable resource to examine cancer metabolic alterations and to identify potential markers of disease. Protocols commonly used for liquid-chromatography mass spectrometry (LC-MS)-based FFPE metabolomics have not been optimized for lipidomic analysis and pre-analytical factors, that potentially affect metabolite levels, were scarcely investigated. We here demonstrate the assessment and optimization of sample preparation procedures for comprehensive metabolomic and lipidomic profiling in FFPE kidney tissue by LC-QTOF-MS. The optimized protocol allows improved monitoring of lipids including ceramides (Cer), glycosphingolipids (GSL) and triglycerides (TAGs) while the profiling capability for small polar molecules is maintained. Further, repeatable sample preparation (CVs < 20%) along with high analytical (CVs < 10%) and inter-day precision (CVs < 20%) is achieved. As proof of concept, we analyzed a set of clear cell renal cell carcinoma (ccRCC) and corresponding non-tumorous FFPE tissue samples, achieving phenotypic distinction. Investigation of the impact of tissue fixation time (6 h, 30 h and 54 h) on FFPE tissue metabolic profiles revealed metabolite class-dependent differences on their detection abundance. Whereas specific lipids (e.g. phosphatidylinositoles, GSLs, saturated fatty acids and saturated lyso-phosphatidytlethanolamines [LPE]) remained largely unaffected (CVs < 20% between groups of fixation time), neutral lipids (e.g. Cer and TAGs) exhibited high variability (CVs > 80%). Strikingly, out of the lipid classes assigned as unaffected, fatty acids 18:0, 16:0 and LPE 18:0 were detectable by high-resolution MALDI-FT-ICR MS imaging in an independent cohort of ccRCC tissues (n = 64) and exhibited significant differences between tumor and non-tumor regions.
    Keywords:  Formalin-fixed paraffin-embedded kidney tissue; Lipidomics; Mass spectrometry; Metabolite extraction; Metabolomics; Non-targeted metabolomics
  7. Rapid Commun Mass Spectrom. 2020 Oct 14. e8977
    Ghosson H, Guitton Y, Ben Jrad A, Patil C, Raviglione D, Salvia MV, Bertrand C.
      RATIONALE: Correct biomarkers determination in metabolomics is crucial for unbiased conclusions and reliable applications. However, this determination is subject to several drifts, e.g. matrix effects and ion suppression in Liquid Chromatography-Mass Spectrometry-based approaches. This phenomenon provokes critical issues for biomarkers determination, particularly during comparative studies dealing with samples exhibiting heterogeneous complexities.METHODS: Occurrence of the issue was coincidentally noticed when studying the environmental impact of a complex bioinsecticide: Bacillus thuringiensis israelensis. Studied samples consisted of insecticide-spiked sediments and untreated control sediments. QuEChERS extractions followed by LC/ESI-Q/ToF analyses were performed on sediments after 15 days of incubation. Meta-metabolomes containing pesticide xenometabolites and sediments' endometabolites were in-depth analyzed using XCMS-based computational data preprocessing. Multivariate statistical analyses (PCA, OPLS-DA) and raw data crosschecks were performed to search for environmental biomarkers.
    RESULTS: Multivariate analyses and raw data crosschecks led to the selection of 9 metabolites as biomarker candidates. However, when exploring mass spectra, co-elutions were noticed between 7 of these metabolites and multi-charged macromolecules originating from the pesticide. Provoked false positives were thus suspected due to a potential ion suppression exclusively occurring in the spiked samples. A dilution-based approach was then applied. It confirmed 5 metabolites as suppressed ions.
    CONCLUSIONS: Ion suppression should be considered as a critical issue for biomarkers determination when comparing heterogeneous metabolic profiles. Raw chromatograms and mass spectra crosscheck is mandatory to reveal potential ion suppressions in such cases. The dilution is a suitable approach to filtrate reliable biomarker candidates before their identification and absolute quantification.
  8. Anal Chem. 2020 Oct 15.
    van der Laan T, Dubbelman AC, Duisters K, Kindt A, Harms AC, Hankemeier T.
      Metabolomics is emerging as an important field in life sciences. However, a weakness of current mass spectrometry (MS) based metabolomics platforms is the time-consuming analysis and the occurrence of severe matrix effects in complex mixtures. To overcome this problem, we have developed an automated and fast fractionation module coupled online to MS. The fractionation is realized by the implementation of three consecutive high performance solid-phase extraction columns consisting of a reversed phase, mixed-mode anion exchange, and mixed-mode cation exchange sorbent chemistry. The different chemistries resulted in an efficient interaction with a wide range of metabolites based on polarity, charge, and allocation of important matrix interferences like salts and phospholipids. The use of short columns and direct solvent switches allowed for fast screening (3 min per polarity). In total, 50 commonly reported diagnostic or explorative biomarkers were validated with a limit of quantification that was comparable with conventional LC-MS(/MS). In comparison with a flow injection analysis without fractionation, ion suppression decreased from 89% to 25%, and the sensitivity was 21 times higher. The validated method was used to investigate the effects of circadian rhythm and food intake on several metabolite classes. The significant diurnal changes that were observed stress the importance of standardized sampling times and fasting states when metabolite biomarkers are used. Our method demonstrates a fast approach for global profiling of the metabolome. This brings metabolomics one step closer to implementation into the clinic.
  9. Anal Chem. 2020 Oct 14.
    Zhu QF, An N, Feng YQ.
      Hydroxy fatty acids are a class of bioactive compounds in a variety of organisms. The identification of hydroxy fatty acids in biological samples has still been a challenge because of their low abundance, high structural similarity, and limited availability of authentic hydroxy fatty acid standards. Here, we present a strategy for the annotation of saturated monohydroxyl fatty acids (OH-FAs) based on the integration of chromatographic retention rules and MS2 fragmentation patterns. Thirty-nine authentic OH-FA standards were used to investigate their retention behavior on a reversed-phase stationary phase (C18) of liquid chromatography, and we found that their retention simultaneously follows two kinds of "carbon number rules". Using the "carbon number rules", the retention index (RI) of all OH-FAs that contain carbon numbers from 8 to 18 (C8-18) can be predicted. Additionally, by studying the MS2 fragmentation of OH-FAs under collision-induced dissociation, we found that the intensity ratio (IR) of the characteristic fragment ions ([M + H]+-63 and [M + H]+-45) is closely related to the position of the hydroxyl group on the OH-FA structure, which is helpful to further identify and confirm the OH-FA isomers. As a result, 97 of 107 potential OH-FAs detected in honey, human serum, and rice seedling by chemical isotope labeling-assisted liquid chromatography-mass spectrometry were annotated upon the RI matching and IR confirming. Furthermore, in order to simplify the annotation process of OH-FAs, we constructed an OH-FA library to facilitate the annotation of OH-FAs. Overall, this study provides a new and promising tool for the in-depth annotation of OH-FA isomers.
  10. Carbohydr Polym. 2020 Dec 15. pii: S0144-8617(20)31187-5. [Epub ahead of print]250 117014
    Šimek M, Lemr K, Hermannová M, Havlíček V.
      The aim of this paper is to review chromatographic and mass-spectrometric methods and underline the best analytical approaches for successful analysis of various hyaluronic acid species in different types of samples. Hyaluronan-degrading enzymes and chemical depolymerization produce di- or oligosaccharides suitable for hyaluronan quantification or structural characterization of hyaluronan derivatives. Efficient purification and pre-column derivatization of hyaluronan disaccharides by reductive amination allow subnanogram quantification in biological samples. The chromatographic separation is capable to distinguish all glycosaminoglycans disaccharides and to resolve hyaluronan fragments with 2-40 monomers. Using electrospray ionization or matrix assisted laser desorption ionization, hyaluronan fragments up to 8 kDa or 41 kDa, respectively, can be observed. One- or two-dimensional chromatographic separation with higly sensitive mass-spectrometric detection is an indispensable tool for revealing substituent position, extent of modification and substitution patterns of chemically modified hyaluronan derivatives. It is essential for studying structure-biological function relationships of hyaluronan and its derivatives.
    Keywords:  Glycosaminoglycans; Hyaluronic acid; Liquid chromatography analysis; Mass spectrometric analysis; Oligosaccharides; Polysaccharide derivative
  11. J Proteome Res. 2020 Oct 15.
    Zhu Y, Wancewicz B, Schaid M, Tiambeng TN, Wenger K, Jin Y, Heyman H, Thompson CJ, Barsch A, Cox ED, Davis DB, Brasier AR, Kimple M, Ge Y.
      Metabolomics-the endpoint of the omics cascade-is increasingly recognized as a preferred method for understanding the ultimate responses of biological systems to stress. Flow injection electrospray (FIE) mass spectrometry (MS) has advantages for untargeted metabolic fingerprinting due to its simplicity and capability for high-throughput screening but requires a high-resolution mass spectrometer to resolve metabolite features. In this study, we developed and validated a high-throughput and highly reproducible metabolomics platform integrating FIE with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS for analysis of both polar and nonpolar metabolite features from plasma samples. FIE-FTICR MS enables high-throughput detection of hundreds of metabolite features in a single mass spectrum without a front-end separation step. Using plasma samples from genetically identical obese mice with or without type 2 diabetes (T2D), we validated the intra and intersample reproducibility of our method and its robustness for simultaneously detecting alterations in both polar and nonpolar metabolite features. Only 5 min is needed to acquire an ultra-high resolution mass spectrum in either a positive or negative ionization mode. Approximately 1000 metabolic features were reproducibly detected and annotated in each mouse plasma group. For significantly altered and highly abundant metabolite features, targeted tandem MS (MS/MS) analyses can be applied to confirm their identity. With this integrated platform, we successfully detected over 300 statistically significant metabolic features in T2D mouse plasma as compared to controls and identified new T2D biomarker candidates. This FIE-FTICR MS-based method is of high throughput and highly reproducible with great promise for metabolomics studies toward a better understanding and diagnosis of human diseases.
    Keywords:  Fourier transform ion cyclotron resonance mass spectrometry; diabetes; flow injection electrospray; high-throughput platform; metabolite fingerprinting; plasma metabolomics
  12. Anal Chem. 2020 Oct 15.
    Greguš M, Kostas JC, Ray S, Abbatiello SE, Ivanov AR.
      In this work, we pioneered a combination of ultralow flow (ULF) high-efficiency ultranarrow bore monolithic LC columns coupled to MS via a high-field asymmetric waveform ion mobility spectrometry (FAIMS) interface to evaluate the potential applicability for high sensitivity, robust, and reproducible proteomic profiling of low nanogram-level complex biological samples. As a result, ULF LC-FAIMS-MS brought unprecedented sensitivity levels and high reproducibility in bottom-up proteomic profiling. In addition, FAIMS improved the dynamic range, signal-to-noise ratios, and detection limits in ULF LC-MS-based measurements by significantly reducing chemical noise in comparison to the conventional nanoESI interface used with the same ULF LC-MS setup. Two, three, or four compensation voltages separated by at least 15 V were tested within a single LC-MS run using the FAIMS interface. The optimized ULF LC-ESI-FAIMS-MS/MS conditions resulted in identification of 2,348 ± 42 protein groups, 10,062 ± 285 peptide groups, and 15,734 ± 350 peptide-spectrum matches for 1 ng of a HeLa digest, using a 1 h gradient at the flow rate of 12 nL/min, which represents an increase by 38%, 91%, and 131% in respective identifications, as compared to the control experiment (without FAIMS). To evaluate the practical utility of the ULF LC-ESI-FAIMS-MS platform in proteomic profiling of limited samples, approximately 100, 1,000, and 10,000 U937 myeloid leukemia cells were processed, and a one-tenth of each sample was analyzed. Using the optimized conditions, we were able to reliably identify 251 ± 54, 1,135 ± 80, and 2,234 ± 25 protein groups from injected aliquots corresponding to ∼10, 100, and 1,000 processed cells.
  13. Ther Drug Monit. 2020 Oct 14.
    Mukai Y, Yoshida Y, Yoshida T, Kondo T, Inotsume N, Toda T.
      BACKGROUND: Recent reports highlight the importance of therapeutic drug monitoring (TDM) of BCR-ABL and Bruton's tyrosine kinase inhibitors (TKIs); thus, large-scale studies are needed to determine the target concentrations of these drugs. TDM using dried plasma spots (DPS) instead of conventional plasma samples is a promising approach. The present study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of BCR-ABL and Bruton's TKIs for further TDM studies.METHODS: A 20-μL aliquot of plasma was spotted onto filter paper and dried completely. Analytes were extracted from two DPS using 250 μL of solvent. After clean-up by supported liquid extraction, the sample was analyzed by LC-MS/MS. Applicability of the method was examined using samples of patient DPS transported by regular mail as a proof-of-concept study. The constant bias and proportional error between plasma and DPS concentrations were assessed by Passing-Bablok regression analysis, and systematic errors were evaluated by Bland-Altman analysis.
    RESULTS: The method was successfully validated over the following calibration ranges: 1-200 ng/mL for dasatinib and ponatinib, 2-400 ng/mL for ibrutinib,5-1000 ng/mL for bosutinib, and 20-4000 ng/mL for imatinib and nilotinib. TKI concentrations were successfully determined for 93 of 96 DPS from clinical samples. No constant bias between plasma and DPS concentrations was observed for bosutinib, dasatinib, nilotinib, and ponatinib, whereas there were proportional errors between the plasma and DPS concentrations of nilotinib and ponatinib. Bland-Altman plots revealed that significant systematic errors existed between both methods for bosutinib, nilotinib, and ponatinib.
    CONCLUSIONS: An LC-MS/MS method for the simultaneous quantification of six TKIs in DPS was developed and validated. Further large-scale studies should be conducted to assess the consistency of concentration measurements obtained from plasma and DPS.
  14. Anal Bioanal Chem. 2020 Oct 15.
    Salomone A, Di Corcia D, Negri P, Kolia M, Amante E, Gerace E, Vincenti M.
      Detection of new psychoactive substances and synthetic opioids is generally performed by means of targeted methods in mass spectrometry, as they generally provide adequate sensitivity and specificity. Unfortunately, new and unexpected compounds are continuously introduced in the illegal market of abused drugs, preventing timely updating of the analytical procedures. Moreover, the investigation of biological matrices is influenced by metabolism and excretion, in turn affecting the chance of past intake detectability. In this scenario, new opportunities are offered by both the non-targeted approaches allowed by modern UHPLC-HRMS instrumentation and the investigation of hair as the matrix of choice to detect long-term exposure to toxicologically relevant substances. In this study, we present a comprehensive and validated workflow that combines the use of UHPLC-QTOF-HRMS instrumentation with a simple hair sample extraction procedure for the detection of a variety of fentanyl analogues and metabolites. A simultaneous targeted and untargeted analysis was applied to 100 real samples taken from opiates users. MS and MS/MS data were collected for each sample. Data acquisition included a TOF-MS high-resolution scan combined with TOF-MS/MS acquisition demonstrating considerable capability to detect expected and unexpected substances even at low concentration levels. The predominant diffusion of fentanyl was confirmed by its detection in 68 hair samples. Other prevalent analogues were furanylfentanyl (28 positive samples) and acetylfentanyl (14 positive samples). Carfentanil, methylfentanyl, and ocfentanil were not found in any of the analyzed samples. Furthermore, the retrospective data analysis based on untargeted acquisition allowed the identification of two fentanyl analogues, namely β-hydroxyfentanyl and methoxyacetylfentanyl, which were not originally included in the panel of targeted analytes.
    Keywords:  Fentanyl; Hair; NPS; NSO; QTOF; SWATH; Untargeted