Showing papers by "Applied Biosystems published in 2009"
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TL;DR: A single-cell digital gene expression profiling assay with only a single mouse blastomere is described, which detected the expression of 75% more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads.
Abstract: Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
2,659 citations
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TL;DR: The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.
Abstract: 9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.
2,336 citations
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TL;DR: The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.
1,194 citations
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TL;DR: Synthesis and cleavage of 10 peptides demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides and assessed the relative effectiveness of various scavengers in suppressing side reactions.
Abstract: The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.
690 citations
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TL;DR: It is concluded that investigators can use TaqMan assays for the selected AIMs as a simple and cost efficient tool to control for differences in continental ancestry when conducting association studies in ethnically diverse populations.
Abstract: To provide a resource for assessing continental ancestry in a wide variety of genetic studies, we identified, validated, and characterized a set of 128 ancestry informative markers (AIMs). The markers were chosen for informativeness, genome-wide distribution, and genotype reproducibility on two platforms (TaqMan assays and Illumina arrays). We analyzed genotyping data from 825 subjects with diverse ancestry, including European, East Asian, Amerindian, African, South Asian, Mexican, and Puerto Rican. A comprehensive set of 128 AIMs and subsets as small as 24 AIMs are shown to be useful tools for ascertaining the origin of subjects from particular continents, and to correct for population stratification in admixed population sample sets. Our findings provide general guidelines for the application of specific AIM subsets as a resource for wide application. We conclude that investigators can use TaqMan assays for the selected AIMs as a simple and cost efficient tool to control for differences in continental ancestry when conducting association studies in ethnically diverse populations.
503 citations
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TL;DR: Reprogramming of advanced epiblast cells from embryonic day 5.5–7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF–STAT3 signalling is shown and reversion of established EpiSCs to rESCs is reported.
Abstract: The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency.
389 citations
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TL;DR: Development in quantitative MS, through the application of stable isotope labelling and scanning techniques, such as multiple reaction monitoring (MRM), have greatly enhanced both the specificity and sensitivity of MS-based assays to the point that they can rival immunoassay for some analytes.
337 citations
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McGill University1, Institute for Systems Biology2, University of British Columbia3, National Institutes of Health4, Invitrogen5, Allergan6, Northeastern University7, Ruhr University Bochum8, Massachusetts Institute of Technology9, Discovery Institute10, Fred Hutchinson Cancer Research Center11, Georgetown University12, University of Gothenburg13, Harvard University14, Thermo Fisher Scientific15, Laval University16, Walter and Eliza Hall Institute of Medical Research17, University of Toronto18, Scripps Research Institute19, University of Alberta20, University of California, Los Angeles21, University College Dublin22, University of Michigan23, University of Pittsburgh24, University of Victoria25, University of Western Ontario26, Wistar Institute27, Yamaguchi University28, Yonsei University29, Agilent Technologies30, Applied Biosystems31, Waters Corporation32
TL;DR: Central analysis determined missed identifications, environmental contamination, database matching and curation of protein identifications as sources of problems in liquid chromatography–mass spectrometry–based proteomics.
Abstract: We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.
324 citations
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TL;DR: Laser microdissection is used to isolate single DA neurons from the substantia nigra pars compacta of controls and subjects with idiopathic Parkinson's disease matched for age and postmortem interval followed by microarrays to analyse gene expression profiling, which provide a 'molecular fingerprint identity' of late-stage Parkinson's Disease DA neurons that will advance understanding of the molecular pathology of this disease.
Abstract: Parkinson’s disease is caused by a progressive loss of the midbrain dopamine (DA) neurons in the substantia nigra pars compacta. Although the main cause of Parkinson’s disease remains unknown, there is increasing evidence that it is a complex disorder caused by a combination of genetic and environmental factors, which affect key signalling pathways in substantia nigra DA neurons. Insights into pathogenesis of Parkinson’s disease stem from in vitro and in vivo models and from postmortem analyses. Recent technological developments have added a new dimension to this research by determining gene expression profiles using high throughput microarray assays. However, many of the studies reported to date were based on whole midbrain dissections, which included cells other than DA neurons. Here, we have used laser microdissection to isolate single DA neurons from the substantia nigra pars compacta of controls and subjects with idiopathic Parkinson’s disease matched for age and postmortem interval followed by microarrays to analyse gene expression profiling. Our data confirm a dysregulation of several functional groups of genes involved in the Parkinson’s disease pathogenesis. In particular, we found prominent downregulation of members of the PARK gene family and dysregulation of multiple genes associated with programmed cell death and survival. In addition, genes for neurotransmitter and ion channel receptors were also deregulated, supporting the view that alterations in electrical activity might influence DA neuron function. Our data provide a ‘molecular fingerprint identity’ of late‐ stage Parkinson’s disease DA neurons that will advance our understanding of the molecular pathology of this disease.
314 citations
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TL;DR: It is shown that miRNA expression profiles are ALL subtype-specific rather than linked to the differentiation stadium associated with these subtypes.
Abstract: MicroRNAs (miRNAs) control the expression of protein-coding genes in normal hematopoietic cells and, consequently, aberrant expression may contribute to leukemogenesis. To identify miRNAs relevant to pediatric acute lymphoblastic leukemia (ALL), we cloned 105 known and 8 new miRNA genes expressed in patients' leukemia cells. Instead of known miRNA genes, new miRNA genes were not evolutionarily conserved. Quantification of 19 selected miRNA genes revealed an aberrant expression in ALL as compared with normal CD34+ cells (P
198 citations
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TL;DR: A robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 μl of serum is developed and optimized, and results showed good correlation with existing ELISA tests applied to the same samples.
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TL;DR: Genotypes from Human Genome Diversity Panel populations were used to further evaluate a 93 SNP AIM panel, a subset of the 128 AIMS set, for distinguishing continental origins and provide additional support for using the 93 AIM set to efficiently identify continental subject groups for genetic studies, to identify study population outliers, and to control for admixture in association studies.
Abstract: Background
Case-control genetic studies of complex human diseases can be confounded by population stratification. This issue can be addressed using panels of ancestry informative markers (AIMs) that can provide substantial population substructure information. Previously, we described a panel of 128 SNP AIMs that were designed as a tool for ascertaining the origins of subjects from Europe, Sub-Saharan Africa, Americas, and East Asia.
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University of Colorado Denver1, University of California, San Diego2, University of California, Irvine3, National Institutes of Health4, Scripps Research Institute5, University of Massachusetts Amherst6, Colorado School of Public Health7, Indiana University8, Max Delbrück Center for Molecular Medicine9, Academy of Sciences of the Czech Republic10, Applied Biosystems11, McGill University12, Royal Prince Alfred Hospital13, QIMR Berghofer Medical Research Institute14, University of Queensland15
TL;DR: The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans and suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.
Abstract: We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.
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TL;DR: Both GC-MS and iTRAQ-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.
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TL;DR: This work shows that the EspC substrate interacts with Rv3868, a cytosolic AAA ATPase, through its C‐terminus, and suggests that proper functioning of the ESX‐1 pathway requires the interaction of multiple ESX-1 substrates and components prior to their secretion.
Abstract: The ESX-1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT-6/CFP-10 dimer, is targeted for secretion via a C-terminal signal sequence on CFP-10 that is recognized by the cytosolic ATPase, Rv3871. However, the selection determinants for other ESX-1 substrates appear to be more complex. Some substrates, such as ESAT-6, are secreted despite lacking signal sequences. Furthermore, all substrates require targeting of the other ESX-1 secreted proteins, a distinguishing feature of this system. How ESX-1 substrates are selected and the basis for co-dependent secretion is unknown. Here we show that the EspC substrate interacts with Rv3868, a cytosolic AAA ATPase, through its C-terminus. Swapping the C-termini of EspC and CFP-10 revealed that these signals are functionally distinct, suggesting that the proteins are targeted via interactions with different ATPases. Surprisingly, biochemical purification experiments demonstrate that these substrates and ATPases form multi-protein complexes inside the cell and identified a new secreted substrate. By interfering with this protein interaction network, we have partially uncoupled co-dependent substrate secretion. Our results suggest that proper functioning of the ESX-1 pathway requires the interaction of multiple ESX-1 substrates and components prior to their secretion. Ultimately, understanding the details of ESX-1 targeting may allow for engineering of better vaccines to prevent tuberculosis.
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TL;DR: The finding of three Y-STR mutations in one father–son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-strs in paternity testing and pedigree analyses.
Abstract: The Y-chromosomal short tandem repeat (Y- STR) polymorphisms included in the AmpFlSTR® Yfiler® polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730-1,764 DNA- confirmed father-son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates
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TL;DR: This study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes.
Abstract: Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes Ago2 was deleted specifically in the growing oocytes Although the Ago2-deficient oocytes are able to develop to mature oocytes, they have abnormal spindles and chromosomes that are unable to cluster together properly This phenotype is very similar to the phenotype of Dicer-deficient oocytes We examined the microRNA expression profile in the Ago2-deficient oocyte and found that the expression of most microRNAs was reduced by more than 80% To determine the downstream genes that are regulated by Ago2, we used microarray analysis on Ago2-deficient oocytes and found that 512 genes were upregulated and 1,073 genes were downregulated (FC > 2, P < 005) Our study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes
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TL;DR: To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants, demonstrating that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
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TL;DR: Recent progresses in characterizing the role of miRNAs in the maintenance and development of ESCs are reviewed.
Abstract: The unique properties of embryonic stem cells (ESCs) to self-renew indefinitely or to differentiate to any cell type have great potential for clinical applications in regenerative medicine. MicroRNAs (miRNAs) are emerging as important regulators of post-transcriptional gene expression and have been implicated as crucial elements in regulating ESCs. Here, we review recent progresses in characterizing the role of miRNAs in the maintenance and development of ESCs.
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TL;DR: It is provided definitive evidence that there is downregulation of a select group of microRNAs in tumours meeting Gynaecological Oncology Group criteria for primary peritoneal carcinoma relative to ovarian serous carcinoma.
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TL;DR: A plasmonic nanostructure for enhanced light excitation is described in this article, which consists of a substrate, an adhesion layer disposed on top of the substrate, a surface plasmmon resonance layer, and a cavity that extends into the surface resonance layer.
Abstract: A plasmonic nanostructure for enhanced light excitation is disclosed. The plasmonic nanostructure includes a substrate, an adhesion layer disposed on top of the substrate, a surface plasmon resonance layer, and a cavity that extends into the surface plasmon resonance layer. The surface plasmon resonance layer is configured to concentrate an applied plasmon field to a bottom portion of the cavity.
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TL;DR: AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution, however, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing.
Abstract: The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.
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TL;DR: The molecular response of holm oak to drought stress and its capacity to recover 9-month-old Quercus ilex seedlings were subjected to three treatments for a 14-d period, and changes in the leaf protein pattern in response to drought and recovery treatments were analyzed by using a proteomic approach.
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TL;DR: Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis using formylvaline as the N-terminal amino acid and cleaved from the resin with ethanolamine.
Abstract: Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycar-bony1 method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, U.V. spectroscopy, and analytical high performance liquid chromatography.
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TL;DR: The PrepFiler™ Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples and facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials.
Abstract: The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.
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TL;DR: The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well as mentioned in this paper.
Abstract: The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C(T)) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/microL. In addition, the multiplex assay can detect as little as 25 pg/microL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFlSTR Amplification Kit to obtain interpretable short tandem repeat profiles.
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TL;DR: It is found that ChIP-seq can be used not only to create gene regulatory maps but also to predict molecular interactions and to inform on the mechanisms for common quantitative variation.
Abstract: Background
The forkhead box/winged helix family members FOXA1, FOXA2, and FOXA3 are of high importance in development and specification of the hepatic linage and the continued expression of liver-specific genes.
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TL;DR: An optimized automated PNA synthesis protocol that is easily scaled up to 10-50 mumol scale syntheses on the automated synthesizer (ABI 433A) and provides the highest coupling yields.
Abstract: An optimized automated PNA synthesis protocol is reported. Under optimal conditions the product yield of a test 17-mer PNA is approximately 90%. The average coupling yield is 99.4%. The synthesis strategy is Boc/Z and the deprotected amine is neutralized in situ. The monomers are added in molar excess to HATU and pre-activated for 60 s before delivery to the resin. The concentration of the activated monomers is 0.08 M during the couplings. Heteroselective solvation provides the highest coupling yields. Acetic anhydride is used as capping reagent followed by a piperidine wash. The protocol has been developed in a 5 mumol scale but is easily scaled up to 10-50 mumol scale syntheses on the automated synthesizer (ABI 433A).
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TL;DR: In this paper, double-strand breaks in DNA activate the kinases ATM and ATR, which block entry into mitosis and delay mitotic progression by controlling spindle assembly in Xenopus egg extracts through the phosphorylation of the centrosomal protein CEP63.
Abstract: Double-strand breaks in DNA activate the kinases ATM and ATR, which block entry into mitosis. ATM and ATR also delay mitotic progression by controlling spindle assembly in Xenopus egg extracts through the phosphorylation of the centrosomal protein CEP63, leading to its delocalization from the centrosome.
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24 Jun 2009-Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment
TL;DR: A method for testing synthetic drugs used to adulterate botanical dietary supplements with prohibited synthetic drugs was developed using liquid chromatography-electrospray ionization-tandem mass spectrometry coupled with a linearity ion-trap system in the multiple reaction monitoring (MRM) plus enhanced product ion (EPI) mode.
Abstract: Adulteration of botanical dietary supplements with prohibited synthetic drugs has become a serious problem. In this paper, a method for testing synthetic drugs used to adulterate botanical dietary supplements was developed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) coupled with a linearity ion-trap system in the multiple reaction monitoring (MRM) plus enhanced product ion (EPI) mode. Twenty-three drugs exhibiting various pharmacological effects, comprising blood pressure and lipid-lowering agents, sedative drugs, anti-diabetic drugs, weight-reducing agents and aphrodisiac compounds, were studied. For all drugs, a single transition was monitored using protonated molecules as precursor ions. EPI spectra were stored in a library and recognized by library searching. Several undeclared drugs were identified in herbal remedies, e.g., glibenclamide, sibutramine hydrochloride and sildenafil. Overall, 35 positive samples were found out of a total of 105 botanical dietary supplements tested. The method was selective, sensitive, rapid, high-throughput and reliable.