bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒12‒19
twenty-two papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Anal Bioanal Chem. 2021 Dec 15.
      Chromatographic retention time information is valuable, orthogonal information to MS and MS/MS data that can be used in metabolite identification. However, while comparison of MS data between different instruments is possible to a certain degree, retention times (RTs) can vary extensively, even when nominally the same phase system is used. Different factors such as column dead volumes, system extra column volume, and gradient dwell volume can influence absolute retention times. Retention time indexing (RTI), routinely employed in gas chromatography (e.g., Kovats index), allows compensation for deviations in experimental conditions. Different systems have been reported for RTI in liquid chromatography, but none of them have been applied to metabolomics to the same extent as they have with GC. Recently, a more universal RTI system has been reported based on a homologous series of N-alkylpyridinium sulfonates (NAPS). These reference standards ionize in both positive and negative ionization modes and are UV-active. We demonstrate the NAPS can be used for retention time indexing in reversed-phase-liquid chromatography-mass spectrometry (RP-LC-MS)-based metabolomics. Having measured >500 metabolite standards and varying flow rate and column dimension, we show that conversion of RT to retention indices (RI) substantially improves comparability of retention information and enables to use of RI for metabolite annotation and identification. Graphical Abstract.
    Keywords:  Metabolite annotation; Metabolomics; Retention time indexing; Reversed-phase
  2. Biomed Chromatogr. 2021 Dec 16. e5299
      Bioanalysis of an endogenous compound such as leucovorin is never an easy task on liquid chromatography tandem mass spectrometer (LC-MSMS). Unless necessary, regulatory guidance discourages to work with surrogate matrix for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography-tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards i.e. leucovorin D4 and 5- methyl tetrahydrofolic acid 13 C5 were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid phase extraction from plasma followed by reverse phase gradient chromatography and mass spectrometry detection with a total run time of 5 minutes. The method was developed and validated from 5-2202 ng/ml for leucovorin and 5-1300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recovery for leucovorin and 5-methyl tetrahydrofolic acid was found to be 100.4% and 100.9 % respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentration of leucovorin and 5-methyl tetrahydrofolic acid and ranged between 651ng/ml to 883 ng/ml and 518 ng/ml to 635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid ranged between 9.3 to 10.5 and 9.2 to 17.6 hours, respectively.
    Keywords:  Bioequivalence; Human Plasma; LC-MS/MS; Leucovorin
  3. Methods Mol Biol. 2022 ;2437 197-213
      The ability to study and visualize metabolites on a cellular and sub-cellular level is important for gaining insights into biological pathways and metabolism of multicellular organisms. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool for metabolomics experiments due to its high sensitivity and small sampling size. The spatial resolution in MALDI-MSI is mainly limited by the number of molecules available in a small sampling size. When the sampling size is low enough to achieve cellular or subcellular spatial resolution, signal intensity is sacrificed making poorly ionized metabolites difficult to detect. To overcome this limitation, on-tissue chemical derivatization reactions have been used to enhance the desorption/ionization efficiency of selected classes of compounds by adding a functional group with a permanent positive charge or one that can be easily ionized. By utilizing several chemical derivatizations in parallel, metabolite coverage can be drastically improved. This chapter outlines methodology for sample preparation and data analysis for on-tissue chemical derivatization using various derivatization reagents.
    Keywords:  Mass spectrometry imaging; Matrix-assister laser desorption/ionization; Metabolite coverage; Metabolomics; On-tissue derivatization
  4. Rapid Commun Mass Spectrom. 2021 Dec 15. e9242
      RATIONALE: Saliva has been widely accepted as a more convenient alternative to serum or plasma in the field of clinical diagnosis. However, the detection of trace components in saliva has been a bottleneck problem. The aim of this work was to develop a highly sensitive and reliable method for simultaneously determining the trace steroid hormones including some with poor ionization efficiency in human saliva by liquid chromatography tandem mass spectrometry (LC-MS).METHODS: Saliva was deproteinated by acetonitrile containing mixed isotope internal standards and extracted by methyl tert-butyl ether. The extraction solution was dried under nitrogen stream and the residue was derivatized by 50 mM O-ethylhydroxylamine hydrochloride in 80% methanol-water solution (v/v). The processed sample was determined by LC-MS in multiple reaction monitoring (MRM) mode.
    RESULTS: The method was successfully established for the simultaneous quantification of seven steroid hormones in human saliva and showed excellent specificity and sensitivity. The quantification limits (LOQs) of all steroid hormones were below 5 pg/ml, especially the LOQ of progesterone was as low as 0.15 pg/ml. The linear correlation coefficients (r) were greater than 0.9990 in the range of 2-200 pg/ml for T, DHEA, A4, P4, P5 and 17OHP4 and in the range of 5-500 pg/ml for 17OHP5. The variability of intra-day and inter-day ranged from 1.86% to 7.83% and 1.95% to 10.4%, respectively. The recovery of the method ranged from 86.9% to 111.1% for all steroid hormones using three spiked concentrations.
    CONCLUSIONS: A novel LC-MS/MS method was developed for simultaneously quantifying seven kinds of trace steroid hormones in human saliva. The results of methodological study showed that the method exhibited excellent sensitivity and reliability for the evaluation of free steroid hormones in human body. It was believed that this method could provide useful information of steroid hormone metabolism for auxiliary diagnosis of some endocrine disorders.
  5. Methods Mol Biol. 2022 ;2437 181-194
      Mass spectrometry imaging (MSI) could provide chemical spatial distribution within a diverse range of samples, but absolute quantitation with those techniques is still challenging. Recent years, ambient liquid extraction-based MSI techniques, such as liquid microjunction surface sampling (LMJSS), have been largely developed and were found to be favorable to quantitation by directly doping standards in the extraction solvent. Here, we describe the detailed experimental protocols and the data processing methods for quantitative MSI with LMJSS. The new methods could have absolute quantitative MSI of both endogenous lipids and small metabolites from tissue samples.
    Keywords:  Calibration methods; Lipidomics; Liquid microjunction surface sampling; Metabolomics; Quantitative mass spectrometry imaging
  6. Methods Mol Biol. 2022 ;2437 117-125
      Nanostructure initiator mass spectrometry (NIMS) with fluorinated gold nanoparticles (f-AuNPs) enables the detection and spatial localization of a breath of polar metabolites and lipids with high spatial resolution and ultrasensitivity. Here we describe the methods and procedures for the synthesis and application of f-AuNPs for NIMS of small molecule metabolites and lipids in biological tissues, encompassing sample preparation, mass spectrometric detection, and data analysis and interpretation.
    Keywords:  Gold nanoparticles; Mass spectrometry imaging; Metabolism heterogeneity; Metabolite imaging; Metabolomics; Nanostructure initiator mass spectrometry
  7. Methods Mol Biol. 2022 ;2437 61-75
      Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods.
    Keywords:  FTICR; FTMS; LAESI; Mass spectrometry; Single-cell analysis; Spatially resolved mass spectrometry; Ultrahigh mass resolution
  8. Drug Discov Today Technol. 2021 Dec;pii: S1740-6749(21)00027-5. [Epub ahead of print]40 69-75
      In this paper, we review the growing development and applications of supercritical fluid chromatography-mass spectrometry (SFC-MS) for the analysis of small molecular analytes and biomarkers in drug discovery. As an alternative chromatographic technique, SFC instrumentation and methodology have dramatically advanced over the last decade. Mass spectrometry (MS) provides the powerful detection capability as it couples with SFC. A growing number of SFC-MS/MS applications were reported over the last decade and the application areas of SFC-MS/MS is rapidly expanding. The first part of this review is devoted to the different aspects of SFC-MS development and recent technological advancements. In the second part of this review, we highlight the recent application areas in pharmaceutical research and development.
    Keywords:  supercritical fluid chromatography, mass spectrometry, SFC-MS, bioanalysis, chiral separation, metabolomics
  9. Methods Mol Biol. 2022 ;2437 3-19
      The unambiguous identification of isobaric (i.e., same nominal mass) and isomeric (i.e., same exact mass) lipids remains a challenging yet vital aspect of imaging mass spectrometry (IMS) workflows. This chapter presents a methodology for the preparation of biological tissue samples and the use of a hybrid mass spectrometer to perform gas-phase charge inversion ion/ion reactions for improved lipid identification. This gas-phase ion/ion reaction method provides lipid structural information beyond what can be obtained via conventional tandem mass spectrometry (MS/MS) experiments. While this procedure is described here for the identification of phosphatidylcholine (PC) analyte cations using 1,4-phenylenedipropionic acid reagent dianions, it can readily be generalized to perform a diverse array of ion/ion reaction chemistries.
    Keywords:  Gas-phase; Imaging mass spectrometry; Ion/ion reactions; Lipid identification
  10. J Pharm Sci. 2021 Dec 14. pii: S0022-3549(21)00690-0. [Epub ahead of print]
      Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used quantitative method in small-molecule pharmacokinetic sample analysis. The linear dynamic range of mass analyzers, typically spanning 3 orders of magnitude, is usually insufficient for this purpose. Utilization of multiple isotopologues has been proposed as a compelling approach to expand the linear dynamic range of LC-MS/MS assays, particularly when the detector is saturated. Isotopologues are a statistical mixture of molecules of the same compound but of different exact masses due to the presence of natural chemical isotopes. While the concept of isotopologues is widely recognized in large-molecule bioanalysis and small-molecule metabolite profiling, it has not been commonly implemented in small-molecule targeted quantification. To increase the awareness of the value of isotopologues in small-molecule LC-MS/MS analysis, this minireview provides the basis of isotopologue distribution in MS/MS and summarizes published studies as well as our own experience in utilizing multiple isotopologues to expand the linear dynamic range of small-molecule LC-MS/MS assays. Considering that utilizing natural isotopologue transitions in the LC-MS/MS assays represents an easy, straightforward, and robust way to expand the linear dynamic range, we believe this method deserves wide application in small-molecule pharmacokinetic sample analysis and can particularly benefit people working in pharmacokinetic labs as well as the GLP bioanalytical labs in pharmaceutical industry.
    Keywords:  LCMS; isotopologues; linear dynamic range; natural isotopologue transitions; small-molecule pharmacokinetics
  11. Methods Mol Biol. 2022 ;2437 77-86
      Over the past couple of years, imaging mass spectrometry (IMS) has arisen as a powerful tool to answer research questions in the biomedical field. Imaging mass spectrometry allows for label-free chemical imaging by providing full molecular information. The IMS technique best positioned for cell and tissue analysis is time-of-flight secondary ion mass spectrometry (ToF-SIMS) because it has the best spatial resolution of all the molecular IMS techniques and can detect many biochemical species and especially lipids with high sensitivity. Because one must rely on the mass and isotopic pattern of an ion in combination with positive correlations with lower mass fragments to help identify its structure, one major problem during ToF-SIMS experiments is the ambiguity when assigning a molecule to a certain mass peak. The solution are instruments with tandem MS capabilities as was already the case for many MALDI-ToF instruments more than a decade ago. It has been a few years since instruments with this capability were introduced and a number of interesting publications have been produced highlighting the advantages in biological SIMS work. Here, we present a protocol describing how tandem MS can be used to elucidate the structure of unknown or ambiguous mass peaks in biological tissue samples observed during ToF-SIMS imaging based on our experiences.
    Keywords:  Fatty acids; Imaging mass spectrometry; Lipids; Orbitrap; SIMS; Tandem MS; Time-of-flight
  12. Plant Cell Physiol. 2021 Dec 17. pii: pcab173. [Epub ahead of print]
      The advancement of metabolomics in terms of techniques for measuring small molecules has enabled the rapid detection and quantification of numerous cellular metabolites. Metabolomic data provide new opportunities to gain a deeper understanding of plant metabolism that can improve the health of both plants and humans that consume them. Although major public repositories for general metabolomic data have been established, the community still has shortcomings related to data sharing, especially in terms of data reanalysis, reusability, and reproducibility. To address these issues, we developed the RIKEN Plant Metabolome MetaDatabase (RIKEN PMM,, which stores mass spectrometry-based (e.g. GC-MS-based) metabolite profiling data of plants together with their detailed, structured experimental metadata, including sampling and experimental procedures. Our metadata are described as Linked Open Data (LOD) based on the Resource Description Framework (RDF) using standardized and controlled vocabularies, such as the Metabolomics Standards Initiative Ontology (MSIO), which are to be integrated with various life and biomedical science data using the World Wide Web. RIKEN PMM implements intuitive and interactive operations for plant metabolome data, including raw data (netCDF format), mass spectra (NIST MSP format), and metabolite annotations. The feature is suitable not only for biologists who are interested in metabolomic phenotypes, but also for researchers who would like to investigate life science in general through plant metabolomic approaches.
    Keywords:  Data sharing; Metabolite profiling; Metabolomics; Plant metabolism; Semantic web
  13. Chem Commun (Camb). 2021 Dec 15.
      Parahydrogen hyperpolarization has been shown to enhance NMR sensitivity in urine analysis by several orders of magnitude if urine samples are prepared by solid phase extraction (SPE). We present a different approach, developed for minimal sample alteration before analysis. Removing SPE from the workflow allows to retain a wider range of metabolites and paves the way towards more universal hyperpolarized NMR metabolomics of low abundance metabolites.
  14. STAR Protoc. 2021 Dec 17. 2(4): 100977
      We describe a protocol for identifying cellular thiol metabolites such as cysteine and cystine in adherent cells using high performance liquid chromatography (HPLC) tandem mass spectrometry-based metabolomics. We applied a modified extraction and sample derivatization protocol to accurately quantify the intracellular levels of labile thiol species and to inhibit oxidation prior to analysis. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020) and Koppula et al. (2021).
    Keywords:  Cancer; Mass Spectrometry; Metabolism
  15. STAR Protoc. 2021 Dec 17. 2(4): 101002
      Here, we present a spatially resolved sampling protocol for the oral human cavity aimed at untargeted metabolomics. We describe the spatial collection of salivary biospecimens, their preparation, and subsequent mass-spectrometry-based untargeted metabolomics analysis. Our protocol avoids complex procedures generally required for gland-specific saliva collection. For the human oral cavity, we provide an easy, flexible, and reproducible solution to comprehensively map the highly heterogeneous environment and elucidate the functionality of salivary components. For complete details on the use and execution of this protocol, please refer to Ciurli et al. (2021).
    Keywords:  Clinical Protocol; Health Sciences; Mass Spectrometry; Metabolism; Metabolomics
  16. J Pharm Biomed Anal. 2021 Dec 07. pii: S0731-7085(21)00636-1. [Epub ahead of print]209 114525
      The global transcription inhibitor terameprocol is being evaluated clinically as an oral formulation to treat high-grade glioma. A sensitive, reliable method was developed to quantitate terameprocol using LC-MS/MS to perform detailed pharmacokinetic studies. Sample preparation involved protein precipitation using acetonitrile. Separation of terameprocol and the internal standard, Sorafenib-methyl-d3, was achieved with a Zorbax XDB C18 column (2.1 × 50 mm, 3.5 µm) and gradient elution over a 2-minute total analytical run time. A SCIEX 4500 or SCIEX 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for terameprocol detection. The assay range of 5-1000 ng/mL was demonstrated to be accurate (92.7-107.4%) and precise (CV ≤ 11.3%). A sample diluted 1:10 (v/v) was accurately quantitated. Terameprocol in plasma has been proven stable for at least 20 months when stored at -70 °C. The method was applied to the measurement of total plasma concentrations of terameprocol in a patient with a high-grade glioma receiving a 300 mg oral dose.
    Keywords:  Assay; Tandem mass spectrometry; Terameprocol; Validation
  17. J Am Soc Mass Spectrom. 2021 Dec 15.
      Current methods typically used for metabolite screening and disease diagnosis often require extensive sample preparation, which increases analysis time and associated costs. While ambient ionization techniques enable the analysis of various samples in complex matrices with little or no sample preparation in a short time (typically within a minute), their reduced selectivity, even when coupled with high-resolution mass spectrometers, limits their application in certain fields. In this study, we have optimized the coupling of paper spray (PS) and leaf spray (LS) ambient ionization techniques with a commercially available ion mobility mass spectrometer (IM-MS) and demonstrated the separation of geometric and constitutional isomers. Ambient ionization techniques allow simultaneous introduction and ionization of samples, while background noise and matrix interference from paper and leaf substrates are filtered out by IM separation, resulting in high sensitivity and selectivity of the PS-IM-MS and LS-IM-MS workflows. In addition, we introduced a novel approach to perform single-field collision cross section (CCS) measurements, which resulted in CCS values that differ by 0.15% and 0.25% from traditional stepped-field and single-field methods, respectively. In addition, we used advanced computational tools to confidently identify analyte structures by comparing CCS values from experimental IM measurements and theoretical calculations. These results suggest that the coupling of ambient ionization methods with ion mobility techniques enables rapid, sensitive, and highly selective analysis that can be used in different fields, such as agrochemical screening and disease diagnostics.
    Keywords:  ambient ionization; direct CCS measurements; ion mobility; mass spectrometry
  18. Drug Discov Today Technol. 2021 Dec;pii: S1740-6749(21)00020-2. [Epub ahead of print]39 81-88
      Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction.
  19. Anal Chem. 2021 Dec 13.
      N6-methyl-2'-deoxyadenosine (m6dA) is a newly discovered DNA epigenetic mark in mammals. N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are common RNA modifications. Previous studies illustrated the associations between the aberrations of these methylated adenosines in nucleic acids and cancer. Herein, we developed Fe3O4/graphene-based magnetic dispersive solid-phase extraction for the enrichment and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) for the measurements of m6dA, m6A, Am, m6Am, and m62A in human urine samples. We found that malic acid could improve the HILIC-based separation of these modified nucleosides and markedly enhance the sensitivity of their MS detection. With this method, we accurately quantified the contents of these modified adenine nucleosides in urine samples collected from gastric and colorectal cancer patients as well as healthy controls. We found that, relative to healthy controls, urinary m6dA and Am levels are significantly lower for gastric and colorectal cancer patients; while gastric cancer patients also exhibited lower levels of urinary m6A, the trend was opposite for colorectal cancer patients. Together, we developed a robust analytical method for simultaneous measurements of five methylated adenine nucleosides in human urine, and our results revealed an association between the levels of urinary methylated adenine nucleosides and the occurrence of gastric as well as colorectal cancers, suggesting the potential applications of these modified nucleosides as biomarkers for the early detection of these cancers.
  20. Methods Mol Biol. 2022 ;2437 253-272
      Mass spectrometry imaging (MSI) data generally contains large sizes and high-dimensional structures due to their inherent complex chemical and spatial information. A variety of data analysis methods have been developed to comprehensively analyze the MSI experimental results and extract essential information. Here, we describe the protocols of data preprocessing and emerging methods for data analyses, including multivariate analysis, machine learning, and image fusion, that have been applied to the data generated from the Single-probe MSI technique. These strategies and methods can be potentially applied to handling data produced from other MSI techniques.
    Keywords:  Image fusion; Machine learning; Multivariate data analysis; Single-probe mass spectrometry imaging
  21. Anal Chem. 2021 Dec 12.
      Endogenous guanidino compounds (GCs), nitrogen-containing metabolites, have very important physiological activities and participate in biochemical processes. Therefore, accurately characterizing the distribution of endogenous GCs and monitoring their concentration variations are of great significance. In this work, a new derivatization reagent, 4,4'-bis[3-(dimethylamino)propyl]benzyl (BDMAPB), with isotope-coded reagents was designed and synthesized for doubly charged labeling of GCs. BDMAPB-derivatized GCs not only promote the MS signal but also form multicharged quasimolecular ions and abundant fragment ions. With this reagent, an isotope-coded doubly charged labeling (ICDCL) strategy was developed for endogenous GCs with high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF MS). The core of this methodology is a 4-fold multiplexed set of [d0]-/[d4]-/[d8]-/[d12]-BDMAPB that yields isotope-coded derivatized GCs. Following a methodological assessment, good linear responses in the range of 25 nM to 1 μM with correlation coefficients over 0.99 were achieved. The limit of detection and the limit of quantitation were below 5 and 25 nM, respectively. The intra- and interday precisions were less than 18%, and the accuracy was in the range of 77.3-122.0%. The percentage recovery in tissues was in the range of 85.1-113.7%. The results indicate that the developed method facilitates long-term testing and ensures accuracy and reliability. Finally, the method was applied for the simultaneous analysis of endogenous GCs in four types of lung tissues (solid adenocarcinoma, solid squamous-cell carcinoma, ground-glass carcinoma, and paracancerous tissues) for absolute quantification, nontargeted screening, and metabolic difference analysis. It is strongly believed that ICDCL combined with isotope-coded BDMAPB will benefit the analysis and study of endogenous GCs.
  22. J Cannabis Res. 2021 Dec 13. 3(1): 50
      BACKGROUND: Pesticide testing for hemp has traditionally focused on techniques like QuEChERS with dSPE and SPE which demand time-consuming sample preparation, typically resulting in poor recovery rates for some pesticides, and requires the use of both LC-MS/MS and GC-MS/MS based instruments to cover the analysis for all regulated pesticides. In this study, we describe a streamlined approach for working with LC-MS/MS featuring a dual electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources using solvent extraction for faster and easier sample preparation and with 80-120% recovery for the analysis of all of 66 pesticides (regulated by California state in cannabis) with low detection limits in hemp.METHODS: A simple solvent extraction with acetonitrile was used to extract pesticides from hemp. A LC-MS/MS system with dual ESI and APCI source was used to determine sensitivity for the analysis of 66 pesticides in hemp matrix, 62 pesticides were analyzed using an 18-min LC-MS/MS method with an ESI source and the other 4 pesticides were measured using a 6-min LC-MS/MS method with an APCI source.
    RESULTS: The limit of quantitation (LOQ) of all 66 pesticides in hemp was in the range of 0.0025-0.1 μg/g which was well below the California state action limits of these analytes in cannabis products. A simple, fast, and cost-effective solvent extraction method was used for sample preparation to get good recovery in the range of 80-120% with RSD less than 20%. The unique ionization mechanism of chlorinated pesticides such as pentachloronitrobenzene using the LC-MS/MS system with an APCI source was elucidated. The proficiency test report generated with the LC-MS/MS method showed acceptable results for all of 66 pesticides in hemp with all of th z scores less than 2 with no false positives and negatives. The stability data collected over 5 days showed RSD less than 20% for 66 pesticides in hemp, and this demonstrated the robustness of the LC-MS/MS system used in this work.
    CONCLUSIONS: A LC-MS/MS method with dual ESI and APCI sources was developed for the analysis of 66 pesticides in hemp. The recovery of all pesticides from a hemp matrix was in the acceptable range of 80-120% with RSD less than 20%.
    Keywords:  Atmospheric pressure chemical ionization source; Electrospray ion source; Hemp; Liquid chromatography-mass spectroscopy; Pesticides