CoSMoS 2013 posters
Displayed from Monday August 12 10:30AM – Wednesday August 14
Authors will be available Monday and Tuesday from 5:00 – 6:30 PM
Case Studies of the Application of Direct Analysis Real Time – Mass Spectrometry to Some Pharmaceutical Analytical Challenges
R. Randy Wilhelm, Shao Huang, Michael Matchett, Cliff Herman
DART Ionization/Desorption was employed in reflection mode directly on the tablets and samples involved in the case studies presented. The DART-SVP interface (IonSense Inc, Saugus, MA) was utilized on the front end of a Thermo LTQ-Mass Spectrometer running Xcalibur (2.01 SP1) software (Thermo Fisher Scientific, San Jose, CA). In one example, tablets of two different formulations of a combination drug were examined in this fashion with no sample preparation. These tablet formulations were compared to a reference formulation to determine which one more closely resembled it. In another case study, black particulate contamination on a trial batch of a prototype dosage form was rapidly identified by direct analysis employing DART-MS, again with no sample preparation. In these case studies, the DART-MS approach proved very successful in achieving the study goals in rapid fashion.
Systems Biology Analysis of the Effects of Acetaminophen Treatment on Energy Metabolism Pathways
Jinchun Sun (1), Yosuke Ando (1,2), Dörthe Ahlbory-Dieker (4), Laura K. Schnackenberg (1), Xi Yang (1), James Greenhaw (1), Lisa Pence (1), Feng Qian (3), William Salminen (1), Donna L. Mendrick (1), and Richard D. Beger (1), Lisette Leonhardt (4), Oliver Schmitz (4), Michael Herold (4)
(1) Division of Systems Biology, NCTR, FDA, Jefferson, AR USA
(2) Medicinal Safety Research Labs, Daiichi Sankyo Co., Ltd., Tokyo, Japan
(3) Z-Tech, Jefferson, AR, USA
(4) metanomics GmbH, Tegeler Weg 33, 10589 Berlin, Germany
Drug-induced hepatotoxicity is one of the major reasons for drug recall and hence it is of major concern to the FDA and consumers. An overdose of the analgesic and antipyretic drug acetaminophen (APAP) can lead to liver failure due to saturation of the normal metabolic pathway. In this case, N-acetyl-p-benzosemiquinone (NAPQI), generated by metabolic oxidation of APAP, can bind irreversibly and covalently to macromolecules. As a consequence, disruption of cell function and necrosis of hepatocytes take place. Although a lot of effort has been put into research over the last 45 years and a lot is known about the toxicity and metabolism of APAP, the community still lacks in a complete mechanistic understanding of APAP-induced hepatic necrosis. Our goal therefore was to comprehensively understand APAP-induced hepatotoxicity regarding its influence on energy metabolism by applying an integrated transcriptomic and multi analytical platform-based metabolomics approach.
Sprague Dawley rats were orally gavaged with different concentrations of APAP. Blood, liver tissue and urine samples were analyzed to evaluate the hepatotoxic effects and impact on energy metabolism at different time points. Open metabolic profiling (by NMR and LC/MS) and broad metabolic profiling (by LC/MS/MS and GC/MS) were applied to examine metabolic changes; 270 metabolites were detected in total from serum/plasma and urine. The metabolic data combined with gene expression profiles of the liver served as a basis for this systems biology investigation of APAP-induced hepatotoxicity. The findings indicate that hepatocyte necrosis is a multistage process that was initiated by GSH-depletion and results from a lack of energy or impaired energy production by mitochondria.
Plant Metabolite Profiling in Crop Trait Research
Philipp Ternes, Nicole Christiansen, Katrin Dietrich, Christoph Edner,
Holger Fahnenstich, Michael Herold
metanomics GmbH, Tegeler Weg 33, 10589 Berlin, Germany
Metabolite profiling (MxP) plays a critical role at each stage of product development within BASF Plant Science. In addition to this “broad profiling” technology, which gives a comprehensive view of the metabolome, targeted methods are available to focus on particular metabolite classes based on project needs. Areas of application include the support of mode-of-action analyses, the use of metabolites as biomarkers, and the analysis of metabolites in the context of the development of compositional traits.
The combination of our “cutting-edge” metabolite profiling platform with versatile data analysis and interpretation approaches creates unique opportunities to advance our projects across the product development pipeline at BASF Plant Science.
As a first example, MxP was applied to investigate physiological base–tip gradients in corn leaves. The results clearly show that such gradients exist and that they are mirrored by transcriptomics and physiological data. As a second example, the technology was applied to investigate the mode-of-action of a new herbicide. Comparison of metabolic signatures showed that its mode-of-action differed from that of previously characterized herbicides. A deeper interpretation of the profiling data suggested a key metabolic step as the potential target site, which was subsequently confirmed by overexpression of a metabolic by-pass.
Two-Dimensional Separation by High Performance Ion Mobility Spectrometry – Liquid Chromatography
Carol Moraff, Amanda Camacho, Eugenie Hainsworth, Clinton Krueger, Anthony Midey, Mark Osgood, and Ching Wu
The first commercially available combined HPLC-HPIMS system was used to perform separations that are difficult by either method alone. Two dimensional retention and drift time plots were produced to demonstrate the advantages of this integrated system.
A combined high performance ion mobility spectrometry (HPIMS) and high performance liquid chromatography (HPLC) system proves to be a powerful tool for chemical analysis. Its advantage comes from the combination of two entirely different separation mechanisms: liquid chromatography separates based on polarity, while ion mobility spectrometry separates based on size and shape. In an integrated HPLC-HPIMS system, a liquid sample separates by polarity in an LC column, and the eluent flows through a line of PEEK tubing to an electrospray ionization needle. The sample is ionized and enters the ion mobility drift tube in the gas phase, where the molecules collide with a drift gas and separate based on size and shape. A Faraday plate detector at the end of the tube creates a signal that is plotted as a two-dimensional retention and drift time plot (RDTP), with LC elution time on the Y-axis and HPIMS drift time on the x-axis. Several mixtures that did not separate by HPLC or HPIMS alone were able to be fully resolved in this system, and their two-dimensional plots give information about their size, shape, and polarity.
Rapid Two-Column Initial Screening in HPLC Method Development
Ken Tseng(1), Toshi Ono(1), Tsunehisa Hirose(2), Kazuhiro Kimata(2)
(1) Nacalai USA Inc. (2)Nacalai Tesque Inc.
An orthogonal two-column initial screening is proposed using a C18 column in water/acetonitrile and a phenyl column in water/methanol. Further refinement of the separation parameters is achieved with applying HPLC columns with unique bonded stationary phases of cholesteryl, naphthalene, and pyrenylethyl functional groups.
A C18 column in water/acetonitrile and a phenyl column in water/methanol are used in the initial screening.
If the initial separation shows promise with the C18 column, the mobile phase is adjusted or the column selection in narrow down further with a C1, C4, C8, or Cholester (cholesteryl functional group) column. The Cholester column exhibits similar hydrophobicity as a typical C18 but it has more shape selectivity. The improved separation on structurally similar compounds is most likely due to the cholesteryl functional group’s relatively rigid structure.
If the phenyl column shows better separation, further mobile phase refinement can be done or other columns with stronger π-π interactions such as π NAP (naphthalene functional group) or PYE (pyrenylethyl functional group) can be tested. The two fused aromatic rings gave the π NAP enhanced π-π interaction. If more is needed for better separation, PYE with its 4-fused aromatic rings provide the strongest π-π interaction. The increased retention on unsaturated or conjugated small molecules could save in the analysis time and ease in the method transfer effort.
Development of a Sensitive Chiral Assay using Supercritical Fluid Chromatography-Mass Spectrometry (SFC-MS/MS) to Evaluate the Metabolic Interconversion of Crizotinib
Amanda King-Ahmad, Thomas McDonald,Chris Holliman
Traditionally, chiral separations have been done using normal phase LC chromatography with chiral selective columns. While this is often effective at separating chiral molecules, there are limitations and assays often have long run times and decreased resolution which in turn drive up the limits of quantitation. In this research, we utilized Supercritical Fluid Chromatography-MS/MS (SFC-MS/MS) to develop a highly sensitive chiral assay to investigate the potential metabolic interconversion of a recently approved cancer treatment. By utilizing SFC, we were able to take advantage of the properties of supercritical CO2 and develop an SFC-MS/MS assay which reduced the LC run time in half, enabled effectively baseline separation of the enantiomers, and allowed for detection down to 1 ng/mL.
Pfizer’s recently approved ALK/MET inhibitor crizotinib (Xalkori®) contains a single chiral center. Crizotinib is the R-enantiomer, whereas the S-enantiomer is approximately 80-fold less active. During the approval process regulatory agencies requested that Pfizer provide experimental evidence that crizotinib does not undergo chiral inversion in vivo to the less active form. It was thought that this evidence could be obtained by generating in vitro interconversion data, circumventing the use of actual human clinical samples and their associated costs and regulatory implications. Since chemically, crizotinib was not expected to undergo interconversion, it was necessary to develop a highly selective chiral assay to separate the two enantiomers and allow for detection of as little as 0.5% conversion in the in vitro studies.
In this research, we utilized SFC chromatography coupled to a triple quadrapole mass spectrometer (SFC-MS/MS) to develop a sensitive assay capable of separating and detecting both crizotinib (R-enantiomer) and its S-enantiomer in in vitro samples. This assay allowed for detection down to 1 ng/mL for both forms using a 7 minute cycle time. This method was easily modified to mitigate the effects of a matrix interference observed in the human hepatocyte samples. The use of this SFC-MS/MS assay allowed us to demonstrate that crizotinib does not undergo significant metabolic interconversion in the human in vitro systems.
Capacity Factor-Based Determination of LogD by High Performance Liquid Chromatography
Log D determination of macrocyclic molecule by using HPLC
Software System for Automated Pesticide Screening
Tim Croley;1 Igor Teslya;2 Graham A. McGibbon;2 Ann M. Knolhoff;1 Scott MacDonald;2 John Callahan;1 Richard Lee2
1 FDA/CFSAN, College Park, MD, USA
2 Advanced Chemistry Development, Toronto, Canada
The importance of a safe food supply has prompted increasing efforts to perform analytical testing for a wide range of potential chemical contaminants. While compounds such as pesticides or toxins are of certain interest, utilizing liquid chromatography with high resolution mass spectrometry to acquire accurate mass full-scan data also has the potential to permit targeted and non-targeted screening from a single acquisition. Sufficient resolution and accuracy mean the data can be correlated with the masses of potential contaminants, allowing for the identification of both target compounds and compounds that are not on a target list (non-targets). Since the data size makes manual processing challenging, the creation and testing of an automated software approach for LC/MS-based pesticide screening is of interest.
This work describes a post-acquisition software system designed for that purpose based on ACD/Labs Spectrus platform.
On-Line coupling of supercritical fluid extraction with supercritical fluid chromatography for chiral separation of racemic mixture containing insoluble impurities
Kyung H. Gahm
Molecular Structure and characterization, Therapeutic Discovery, Amgen Inc
Thousand Oaks, CA
In drug discovery laboratories, chromatographic resolution is routinely used to obtain optically pure active pharmaceutical ingredients (APIs), as different enantiomers may have different pharmaceutical effects. At a discovery stage, it is not uncommon that the synthetic product is a complex mixture containing achiral/chiral impurities, inorganic salts, or metal catalysts, which make direct chromatographic separation impossible without pretreatment. Recrystallization, trituration, or solvent extraction is a common method for sample pretreatment. Normal phase flash chromatography is the most frequently used technique in the discovery laboratories for purification or sample pretreatment before submission for chiral separation.
Recently, we published a purification method for chiral separation with supercritical fluid chromatography (SFC) utilizing solubility determinations in SFC mobile phases. Understanding solubility variations in diverse supercritical fluid media is important to increase purification throughput [1-2]. Differential solubility enhancement of a compound (or impurity) under supercritical fluidic conditions can make supercritical fluid extraction (SFE) an improved alternative approach to clean up samples before chromatographic purification.
In this presentation, the setup of an on-line coupled SFE/SFC system will be described. The advantages of this system will be discussed using examples of chiral separations of a racemic mixture containing insoluble impurities. The system was evaluated for preparative separations employing stacked injections of SFE extractant into the SFC system with various injection durations to maximize column loadability.
 K. Gahm, H. Tan, J. Liu, W. Barnhart, J. Eschelbach, S. Notari, S. Thomas, D. Semin, J. Cheetham, J. Pharm. and Biomed. Anal., 2008, 46, 831.
 K.Gahm, K. Huang, W. Barnhart, W. Goetzinger, Chirality, 23, E65-73, 2011 DOI:10.1002/chir.20967.
Development on Analytical method for HPLC Fingerprinting of Berberis
Daya Bhardwaj, Rishu Kalra , and Nutan Kaushik
TERI, New Delhi
We have optimized method for extraction and HPLC fingerprinting of Berberis aristata species using various combinations of the solvents.
No comprehensive work on the fingerprinting of Berberis aristata is on record presently, hence this study we explored the new methods for quality evaluation of medicinal plant Berberis aristata and its related species used extensively due to its various medicinal properties. Method of extraction , solvent , sample to solvent ratio and HPLC fingerprinting method has been standardized
How can we get data for “all metabolites” in Chlamydomonas reinhardtii ?
Nutan Kaushik , Tobias Kind, William Wikoff,Zipora Tietel, Oliver Fiehn
UC Davis Genome Center, Davis, CA
Chlamydomonas reinhardtii is a single celled algae belonging to the division of Chlorophyta . It is highly adaptable and can survive in many different environments throughout the world. It is a candidate species for production of products for biofuel uses. Beside lipids and primary metabolites, C. reinhardtii is reported to produce variety of specialized compounds such as xanthophylls, carotenoids, steroids, alkaloids, flavonoids. But how can we obtain trustworthy annotations for as many small molecules with as few analytical techniques as possible? We here present methods for combining different extraction methods and LC-QTOF MS and GC-TOF MS profiling of the C. reinhardtii metabolome. We discuss results for compound annotations using a variety of mass spectral libraries, including the new virtual MS/MS library LipidBlast.
Evaluation of 25 Chiral stationary phases using Waters UPC2 to identify the best screening system for chiral compounds
Mengling Wong and Chris Hamman
1) Evaluated 25 CSPs with 80 compounds (40 commercially available, 40 proprietary)
2) We were able to evaluate the 25 CSPs due via a fast method we developed
3) The fast method worked well because the UPC2 is well engineered
4) The algorithm allowed us to go through the over 500,000 combos of columns to boil it down to the two best “teams.” Team one separated (R>= 0.3) 98% of the 80 compounds.
It is well known that racemate compounds need to be separated into each enantiomer due to the fact that each will have different biological activity. One enantiomer may be therapeutic and the other may cause an adverse effect. The challenges we face in our Discovery Chemistry Purification group is the number of compounds being submitted that have at least one chiral center. It is the purification group’s responsibility to separate the enantiomers of highest purity and recovery. Moreover, we want to analyze and separate the chiral compounds in a fast and reasonable time. Here we will show how we use the Waters UPC2 instrument to screen racemic compounds using a gradient to sequentially screen each column. After analyzing commercial and internal compounds of basic, acidic, and neutral molecules we have selected 18 chiral stationary phases that meet our needs. These columns are divided into 3 tiers. 95% of the chiral separation are done on the first tier which include AD, Cel-1, Cel-4, ID, Whelko-1, and AS.
Automating High Resolution LC/MS Mass Re-Calibration for Improved Accuracy and Reduced False Positive Results
Mark A. Bayliss1; Stephane Murphy1; Joseph Simpkins1; Ann Knolhoff2; Timothy Croley2; John Callahan2
1 Virscidian Inc, Cary, NC
2 FDA, Center for Food Safety & Applied Nutrition, College Park, MD
Both targeted and non-targeted full scan MS assays rely on the ability to extract and process mass spectra resulting in extracted ion chromatograms (EIC). Data processing in almost all commercial and in-house developed data processing packages requires a user to manually enter data extraction parameters that are then used in the data reduction and this includes the windows for target masses. In most cases data extraction is reduced to a series of fixed values that are applied irrespective of the mass being evaluated, leading to a compromise in the specificity of data extraction. This presentation focuses on recent developments to automate the determination of the primary influencing variables in data extraction and then apply this knowledge to automatically re-calibrate the mass scale on a run to run basis without the need for added mass calibration standards or manually entered tolerance windows.
Extraction-Injection in preparative scale SFC
Mohamed Shaimi and Geoffrey B Cox
PIC Solution SAS,
PIC Solution Inc
The issues around sample introduction in preparative scale supercritical fluid chromatography (SFC) have long been a subject of discussion. Recent papers have discussed the theoretical features of mixed stream and of modifier stream injections have contributed to this (1,2).
Mixed stream injection, where the sample is first dissolved in the polar modifier and injected into the mobile phase (ie the CO2 – modifier mixture), has similar features to those seen in normal phase preparative HPLC where the sample is similarly dissolved in a polar solvent (usually it is not soluble in the low polarity component) and injected into the mixed mobile phase. Here the sample band is distorted to a greater or lesser extent due to the local strongly eluting conditions in the sample band before and as it mixes with the lower polarity mobile phase. This usually results in distortion at the leading edge of the band, where the sample concentration is highest and therefore has significant influence on the production rate attainable from the system. The effects diminish at high modifier concentrations as the sample solvent approaches the mobile phase in chromatographic strength.
An alternative approach is to dissolve the sample in the modifier but then to introduce it into the modifier stream before it is mixed with the non-polar mobile phase constituent. This has the effect of making no change in the mobile phase composition during the injection, ensuring no peak distortion due to the injection solvent. At lower flow rates, however, this technique leads to band broadening simply by the lower flow rate of the modifier stream; at 10% modifier concentration a 1ml injection volume at a total flow rate of 100 ml/min will expand to an injected band of 10 ml in the column. The technique further requires that the mobile phase is adequately mixed downstream of the injection device and that the total hold-up volume post injection is small.
Both injection techniques suffer from a further problem in that the sample is always introduced as a concentrated solution in the modifier. If the solubility in the mixed mobile phase is lower than that in the modifier, there is a strong possibility that some of the injected material will be precipitated on mixing with the mobile phase. If the solubility in the modifier is lower than in the mixed stream, a certain percentage of the production rate will be lost and the sample loading will be reduced relative to an injection with the mobile phase as sample solvent.
Data was previously presented (3) on an approach to eliminate the problems associated by the introduction of the sample in a solution in the modifier by dissolving it directly in the supercritical mobile phase. The present work is an extension of this original idea and describes the performance of an injection system relying on dissolution of the sample in the supercritical fluid mobile phase prior to introduction to the column and comparing the performance with that of injection in the mixed mobile phase from a modifier solution as well as injection into the modifier stream. Besides the advantage of the elimination of the precipitation of sample as it meets the mobile phase stream which is a continuing difficulty in preparative scale SFC, injection by extraction of the solid sample by the supercritical stream often allows a higher solubility than in the modifier alone and also, because of the elimination of the band deformation resulting from the other techniques, allows a higher productivity from the preparative system. Further advantages are that it is easy to control the injection temperature, also allowing a higher sample concentration than injection from a solution at room temperature, and that the material in the sample which is insoluble in the mobile phase remains in the sample container rather than being filtered out in the column inlet frit. The cleaner injected material is expected to improve column lifetime.
1. Keng Hong Goh and Arvind Rajendran, A study of injection strategies on separation efficiency and productivity in preparative supercritical fluid chromatography, PREP 2011 conference, Boston, MA, July 2011.
2. Arvind Rajendran, Large Volume Injections in SFC: Experiments and Theory, SFC 2012, Brussels, Belgium, October 2012.
3. M Shaimi, Direct sample injection technique for preparative supercritical fluid chromatography: solventless injection. 14th International Symposium on preparative/process chromatography, PREP 2001, Washington, DC., May 2001
Mohamed Shaimi and Geoffrey B Cox
PIC Solution SAS
PIC Solution Inc
The use of mass spectrometric detection is of increasing importance in SFC, for both analytical and preparative separations as the technique is extended beyond the chiral applications for which the technique continues to be widely used. This poster describes work directed to the interface of a single quadrupole mass spectrometer with both analytical and preparative SFC systems with a focus on maintaining simplicity with function.
In an analytical configuration, the entire column flow from a 4.6 mm id column cannot be handled by the mass spectrometer and it is necessary to split the flow from the column. In this case the simplest interface involving splitting the column effluent between the back pressure regulator and a capillary of suitable dimensions leading to the MS detector is the most useful. An advantage of SFC over HPLC is the high velocity of the mobile phase mixture as it is depressurized as it passes through the capillary; the delay between the UV detector response (upstream of the split) and that of the MS is hard to measure. The APCI interface was seen to be superior to the electrospray in sensitivity and ease of use as no additive to enhance detector response was needed. In neither case, interestingly enough, was the most abundant ion the (perhaps naively) expected M+1 species. Guaiphenesin, for example, picks up a molecule of methanol (the co-solvent) to give a M+31 species while trans-stilbene oxide undergoes a more complicated process to arrive at a M+14 species. In the electrospray source, the most abundant ions are the sodium adducts despite the inclusion of a proton source (formic acid) in the mobile phase.
In the case of preparative chromatography, the critical factor is detector overload. Using the same interface as in the analytical example at preparative loads in a SFC-PICLab PREP 100 system saturated the detector for approximately 15 minutes! Thus, it is important to dilute the flow into the mass spectrometer. A 1:20 split using a cycling splitter valve feeding a 0.5 ml/min make-up flow was found to be appropriate for the separations studied (although this certainly needs to be variable, depending on the ease of ionization of the products). Given the short delay time between the column outlet and the mass spectrometer detector, it is also important to have a fast transit through the collection system; using a gas-liquid separator at the point of collection greatly facilitates the collection as the product reaches the separator at approximately the same point in time as it reaches the mass spectrometer. Again, the APCI source was seen to be simplest to use, allowing collection to be set up using the M+1 ion rather than the M+23 ion for the EI interface and avoiding the use of formic acid (or similar) in the make-up flow leading to the detector.
A workflow for leachable & extractable analyses of medical IV bag using high resolution LCMS, GCMS, and ICPMS
Kate Comstock, Ekong Bassey, John Schmelzel
Thermo Fisher Scientific
LCMS, GCMS, and ICPMS are major techniques for L&E analyses: LCMS targets medium to high molecular weight species and GCMS targets semi-volatile and volatile species. ICPMS analyzes metal elements.
This study presents a comprehensive workflow for L&E analysis by analyzing IPA extract of commercial IV bags using Thermofisher Scientific Q Exactive™ benchtop Orbitrap mass spectrometer, Thermo Scientific ISQ single Quadrupole GCMS, and Thermo Scientific* iCAP Q ICP-MS.
In this study, a commercial IV bag was filled with IPA at room temperature. The samples were taken at 30 minutes and 1 hour respectively. The samples were analyzed by LCMS system consisting Ultimate3000 LC and Q Exactive MS. Full scan MS and data-dependent top-3 ms/ms were acquired at HR 70000/3500 (FWHM).
The IPA extracts from commercial IV bag were analyzed by HR-LCMS, GCMS, and ICPMS. The LC chromatographic separations were conducted on an Acclaim C18 (2.1x 150 column 2.2 µm). The HRAM MS data were processed with Thermo Scientific software SIEVETM and Mass FrontierTM for components extraction and structure elucidation. The preliminary data shown that DEHP was the major component, others including antioxidants and breakdown polymers.
Multiple Dissociation Techniques for Comprehensive API Impurity Profiling
Thermo Fisher Scientific
Commercial compound Adefovir Dipivoxil ( from Sigma-Aldrich) was used in this study. Sample (0.5 mg/mL in ACN:H2O at pH 4.5) was chromatographically separated by UHPLC (Accela, Thermo Fisher Scientific) on a Hypersil GOLD C18 column (2.1×150 3µm) with a mobile phase composed of A: Water, B:ACN and C: 100 mM ammonium acetate (pH 5) with gradient. The LC eluent was introduced to a Velos Pro linear trap mass spectrometer equipped with a HESI II ion source. Positive ion mode full scan MS and MSn data was collected in a data-dependent fashion by utilizing combinations of both CID and trap HCD dissociation technologies. The MS data were processed using Mass Frontier software for component identification and structural elucidation.
The combination of two fragmentation technologies takes advantage of the unique capabilities of both CID and HCD to obtain rich information for impurity identification. Trap HCD generates more fragment ions compared with CID at the same NCE level, it also has broad mass range which gives low mass fragment ions information, all this provides more structure information. Conversely, CID MSn provides step by step structure linkage which is valuable information for unknown structure elucidation.
The preliminary results in this study demonstrate that this unique workflow, utilizing multiple fragmentation techniques, in combination with post-acquisition process software Mass Frontier, allows streamline impurities identification and structure elucidation, greatly increased the speed and confidence of unknown impurity analysis.