Tskgel® UP-SW3000, 2 Μm U/HPLC SEC Columns

Volume 16 Number 2 May 2018 www.chromatographyonline.com TSKgel® UP-SW3000, 2 μm U/HPLC SEC Columns

Optimized for Size Exclusion Chromatography/Mass Spectrometry (SEC/MS) Analysis

+15 54143 Mass Spectrum 3610.52 100 +16 3384.90 54181 90 80 70 60 +14 +24 50 +29 +17 3868.36 1868.02 2256.95 40 +23 3185.85 30 +31 2356.77

Relative abundance 1748.69 +21 20 2581.06 +18 +13 3008.97 54219 10 4165.78 0 MALDI Imaging MS for 1500 2000 2500 3000 3500 4000 Interdism/zciplinary Research 54086 Bispecific T Cell Engager (BiTE) Deconvoluted Mass Spectrum ApproximateRecent Molecular Advances Mass= 55 kDa in ICP- MS and Applications 53840 54440 55040 55640 Mass (Da)Sheathless CE–MS for Volume-

The TSKgel UP-SW3000, 2 μm SEC column can beRestricted used as a platform Metabolomics method for bispeciﬁ c antibody accurate mass determination using SEC/MS. LC–MS Analysis of Mayonnaise

www.tosohbioscience.com Lipid Oxidation Products

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Column Shedding and Carryover Analysis TSKgel 2 μm, UP-SW3000 Column Characteristics 100

90 Blank before 10 ug of mAb injection 80 Particle size ……………………..2.0 μm 70 Pore size …………………………25 nm 60 Phase chemistry………………...Diol 50

40 pH stability ……………………...2.5-7.5 Relative abundance 30 Calibration range (proteins) ….10-500 kDa 20

10 0.4 3.8 6.5 9.4 12.0 13.9 1221.9938 1221.9926 1221.9908 1221.9919 1221.9930 1221.9871 0 0 2 4 6 8 10 12 14 Retention time (minutes) TSKgel UP-SW3000, 2 μm Columns 100

90 Part number Description ID (mm) Length (cm) Blank after 10 ug of mAb injection 80 23449 TSKgel UP-SW3000 4.6 15 70 23448 TSKgel UP-SW3000 4.6 30 60 TSKgel UP-SW3000 23451 4.6 2 50 DC Guard Column 40

Relative abundance TSKgel UP-SW3000 23450 4.6 2 30 Guard Column 20

10 1.4 2.5 6.6 8.2 12.3 1221.9886 1221.9945 1221.9939 1221.9923 1221.9912 0 0 2 4 6 8 10 12 14 Retention time (minutes) Experimental Conditions Column: TSKgel UP-SW3000, 2 μm, 4.6 mm ID × 30 cm For more information visit Mobile phase: 20 mmol/L ammonium acetate, tosohbioscience.com 10 mmol/L ammonium bicarbonate; pH 7.2 Flow rate: 0.35 mL/min MS instrument: Q Exactive™ Plus HPLC instrument: Nexera® XR UHPLC system Temperature: 30 ºC

TSKgel and Tosoh Bioscience are registered trademarks of Tosoh Corporation. No shedding or carryover was observed via MS Q Exactive is a trademark of Thermo Fisher Scientiﬁc Inc. total ion chromatogram. Nexera is a registered trademark of Shimadzu Corporation.

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MALDI Imaging MS for Interdisciplinary Research Recent Advances in ICP- MS and Applications Sheathless CE–MS for Volume- Restricted Metabolomics LC–MS Analysis of Mayonnaise Lipid Oxidation Products

magentablackcyanyellow ES1048165_LCGCSUPP0518_CV1.pgs 05.01.2018 15:51 ADV magenta black cyan yellow Copyright © 2017 PerkinElmer, Inc. 400376_05 All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. Learn more atwww.perkinelmer.com/gc www.perkinelmer.com/chromsupplies For therightchromatography consumables, goto simply betterforthemostimportantapplicationsofall–those importanttoyou. desorption, andhands-freeliquidorSPMEsampleprep.Highly capableClarussystemsare Our newClarus handle more applications 5WRGTKQTUGPUKVKXKV[ECRCEKV[CPFVJTQWIJRWVsYKVJƃGZKDKNKV[VQ reruns. Andflexible,becauseweintegratebest-in-classTurboMatrix efficient. Consistent,becauseitdeliversprecise,repeatablesample introductionandfewer superfast ovencool-down,andprogrammabletemperatureinjectors makeitmuchmore and moreflexiblethanever.Productive,becauseourproprietary autosamplertechnology, ES1048221_LCGCSUPP0518_CV2_FP.pgs 05.02.2018 19:24 ADV ® 590 and 690 systems are making GC more productive, more consistent – 590and690systemsaremakingGCmoreproductive,consistent – INTOFORM ANART HIGHLY CAPABLE GC WE ’ VE TURNED VE TURNED ® headspace, thermal headspace,thermal HILICpak

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Volume 16 Number 2 May 2018 www.chromatographyonline.com Articles

Recent Advances and Trends in Inductively Coupled Plasma–Mass Spectrometry and Applications 8

V. Balaram There have been exciting recent advances in ICP-MS instrumentation, such as the development of magnetic sector ICP-MS, multicollector ICP-MS, time-of-flight ICP-MS, and triple-quadrupole ICP-MS, as well as developments in the coupling of laser ablation (LA) and laser-induced breakdown spectroscopy (LIBS) to ICP-MS. This article surveys these developments and looks to the future.

The Use of MALDI Imaging Mass Spectrometry for Interdisciplinary Research: From Metabolomics to Pesticides 14

Shannon Cornett, Mike Easterling, and Charles Pineau Matrix-assisted laser desorption–ionization (MALDI) imaging mass spectrometry allows direct, in situ, label-free measurement of proteins, peptides, lipids, small-molecule drugs and their metabolites, and other chemicals in tissues. In a range of applications, the unique information generated by MALDI imaging can make a significant contribution to understanding factors such as molecular and metabolic mechanisms and the transport and localization of compounds or metabolites with human, animal, or plant species.

Resolving Volume-Restricted Metabolomics Using Sheathless Capillary Electrophoresis–Mass Spectrometry 20

Rawi Ramautar Recent advances have significantly improved the performance of capillary electrophoresis–mass spectrometry (CE–MS) for the profiling of polar and charged metabolites in volume-restricted or mass-limited biological samples. Here, those advances are discussed, and attention is also devoted to various technical aspects that still need to be addressed.

Liquid Chromatography–Atmospheric Pressure Photoionization- Mass Spectrometry Analysis of the Nonvolatile Precursors of Rancid Smell in Mayonnaise 24

Boudewijn Hollebrands and Hans-Gerd Janssen For lipid-containing food products like mayonnaise, determining nonvolatile lipid oxidation products, the precursor compounds for rancidity, makes it possible to predict product shelf life at an earlier stage in product development. A method based on normal-phase liquid chromatography with atmospheric pressure photoionization-mass spectrometry (LC–APPI-MS) was developed for this purpose.

Departments

Products ...... 34

Ad Index ...... 38

Cover image courtesy of Charles Pineau of Inserm and the Protim Core Facility at the University of Rennes 1, in Rennes, France

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Recent Advances and Trends in Inductively Coupled Plasma– Mass Spectrometry and Applications

Today, quadrupole inductively coupled plasma–mass spectrometry (ICP-MS) occupies an invaluable position in modern analytical laboratories worldwide because of its multielement and multi-isotope capability, high sensitivity, very limited interference effects, precision, and accuracy. Over the past three decades, however, there have been exciting advances in ICP-MS instrumentation, such as the development of high-resolution or magnetic sector ICP-MS, multicollector ICP-MS, time-of-flight ICP- MS, and triple-quadrupole ICP-MS. In addition, there have been important developments in the coupling of laser ablation (LA) and laser-induced breakdown spectroscopy (LIBS) to ICP-MS. This article surveys these developments and looks ahead to the future.

V. Balaram

ne of the most innovative developments in the area of discrimination (KED) have been introduced. Recently, two Oanalytical instrumentation during the past 50 years is manufacturers have introduced ICP-MS/MS systems (or tri- the development of the quadrupole inductively couple-quadrupole ICP-MS) systems, which further improve on pled plasma–mass spectrometry (ICP-MS) and the subsequent the interference-removal capabilities of collision–reaction developments of magnetic sector and time-of-flight (TOF) cells. In contrast to the traditional quadrupole-based ICP-MS instruments (1–3). Over the past three decades, ICP-MS, with systems equipped with a collision–reaction cell, ICP-MS/MS its excellent detection limits, multielement and multi-isotope is characterized by the presence of two quadrupoles with a capability, limited interferences, and a wide linear dynamic collision–reaction cell in between, which can be operated with range, has become widely accepted as an established technique a variety of cell gases, although one firm uses an octopole col- for trace- and ultratrace-element analysis, with thousands of lision–reaction cell to do the similar function (Figure 1). The instruments in use all over the world not only in the forefront first quadrupole provides a mass-selection step before the of major areas such as earth, environment, ocean, and pharma- cell, which gives better control over the ion–molecule reac- ceutical sciences but also in emerging areas like proteomics and tions taking place in the cell by removing the matrix, followed nanoparticle analysis (4–8). Recent developments in microelec- by the mass selection by the second quadrupole. This double tronics and information processing have immensely contributed mass selection before and after the reaction cell, together with to making these instruments extremely smart, user friendly, and controlled ion–molecule chemistry, enables the determination compact (9). The quest for interference-free determination and of elements difficult to determine without this capability and lower detection limits has driven numerous subsequent develop- delivers more accurate results for complex samples. It also im- ments in recent years, which include coupling of laser-ablation proves detection limits. For example, 129I in soil samples was sampling with laser-induced breakdown spectroscopy (LIBS), determined using ICP-MS/MS, with the objective of investi- collision–reaction cells even in multicollector instruments, and gating radioiodine released by the Fukushima Daiichi Nuclear multiple quadrupole MS arrangements. This article examines Power Plant (FDNPP) accident. High background caused by some of these recent developments in ICP-MS technology that 129Xe impurities in argon plasma gas and polyatomic ions such 127 + 127 + have made considerable impact in science and technology. as IH2 and ID usually make it difficult to determine this isotope using conventional ICP-MS instruments. Oxygen ICP-Tandem Mass Spectrometry was used as a reaction gas to reduce the background intensity To overcome spectral interferences in quadrupole ICP-MS, of m/z 129, principally by 129Xe. Accurate and extremely pre- features such as collision–reaction cells and kinetic energy cise 129I/127I ratios obtained are consistent with those obtained

magentablackcyan ES1048066_LCGCSUPP0518_008.pgs 05.01.2018 15:28 ADV chromatographyonline.com May 2018 Current Trends in Mass Spectrometry 9

Table I: Determination of As (μg/g) in mineral fertilizers and agricultural gypsum by accelerator mass spectrometry (AMS) using single quadrupole and MS/MS mass shift mode (11) (10). In another interesting application, Sample Single Quadrupole MS/MS, 0.50 mL/min O Machado and colleagues (11) have ac- 2 curately determined arsenic using Fertilizer 1 12.8 ± 1.1 8.9 ± 0.7 ICP-MS/MS by applying mass shift re- Fertilizer 2 36.8 ± 2.5 29.2 ± 1.4 action in the collision–reaction cell. Ac- Gypsum 1 7.9 ± 0.04 0.37 ± 0.01 curate determination of 75As+ usually is Gypsum 2 6.4 ± 1.4 0.65 ± 0.04 a challenge because of the well known isobaric overlaps caused by polyatomic species, such as 40Ar35Cl+, originated from the presence of elements of the matrix in the argon plasma. In ICP-MS/MS, Detector 75 + 75 16 + Vacuum As species are converted to As O interface Plasma torch ions by the addition of oxygen gas into the collision–reaction cell, which allows the accurate determination of As

Quadrupole Q2 in samples with high contents of rare Quadrupole Q1 earth elements (REEs), with REE-related interfering species such as 150Nd2+ and Octopole collision–reaction cell 150Sm2+. The results presented in Table

Spray chamber I show that in single-quadrupole mode concentrations are higher than those determined by MS/MS mass shift mode, Figure 1: Schematic diagram of a triple-quadrupole ICP-MS system with a collision–reaction cell and this difference is more pronounced located between two quadrupoles. (Courtesy of Agilent Technologies.) in agricultural gypsum samples, where

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detection of elements such as F, O, H, N, Si, Se, As, Ca, and S very challenging or even impossible with conventional ICP-MS system LA-ICP-MS systems. As a result, other techniques such as X-ray fluorescence (XRF) spectroscopy are being used to Video camera Detector obtain complementary information.

➞ Optical zoom However, cross-correlation between exact sample locations, excitation vol- Laser umes, and resolution (spatial and depth) may vary substantially, leading to faulty results. But tandem LA-LIBS coupled to Particles Spectrometer Emission collection ICP-MS is used to successfully chemi- transport cally correlate, image, and map major, Ablation chamber minor, and trace elements and isotopes through two-dimensional (2D) layer-by- layer and 2D cross-sectional imaging as well as three-dimensional (3D) volume Figure 2: A schematic system of a tandem LIBS-LA-ICP-TOF-MS system (29). reconstruction of the elemental distribu- REE concentrations are up to 10-fold species to create a spectral signature of tion in geological samples (16) (Figure higher than those found in fertilizers. the elements, and the analysis can be 3). By adopting computed tomography ICP-MS/MS also showed greater ability performed under ambient conditions. imaging principles to visualize multi- for the accurate determination of the Typical limits of detection of metallic ple elemental and isotopic distributions fission products 135Cs, 137Cs, and 90Sr, elements reported for LIBS are in mi- in bastnaesite mineral ore matrix, ex- which are radioactive and have detection crograms-per-gram to weight percent panded elemental coverage from a single limits as low as 0.01 pg/mL (12). There levels. LA-ICP-MS, on the other hand, sampling event is possible. In a forensic are several such examples in the recent acquires the mass-to-charge ratios char- application, Subedi and colleagues (17) literature where ICP-MS/MS has been acteristic of the elemental composition used this combination technique to dis- very effectively used for the sensitive of the ejected particles resulting from criminate printing inks with informa- and interference-free determination of the laser–material interaction with de- tion from the atomic or ionic emissions various elements in several matrices (13). tection limits of different elements in and isotopic composition (m/z) by using the range of sub-microgram-per-gram the fingerprint spectra of each ink sam- Tandem LA-LIBS levels. Because isotopic information can ple. Such combination instrumentation Coupled to ICP-MS also be obtained by LA-ICP-MS, the is becoming very popular with several Laser-ablation ICP-MS (LA-ICP-MS) combination of LIBS and LA-ICP-MS such examples having been seen in dif- is a powerful technique for direct solid (Figure 2) can be used to obtain chem- ferent areas of application. analysis with unique capabilities such as ical information (both elemental and microanalysis, depth profiling analysis, isotopic) over a wider concentration Collision–Reaction Cell two-dimensional elemental or isotope range. There are some exciting applica- Multicollector ICP-MS mapping, and the analysis of conduc- tions in the recent literature where si- Nontraditional stable isotopes such as tive and nonconductive materials. An- multaneous tandem LA-LIBS coupled V, Cu, Mo, Ba, K, Pt, Pd, Ag, Ce, Er, other emerging analytical technique to an ICP-MS system had an impact on and Si, with small variations in natu- for direct solid analysis that uses LA, qualitative and quantitative analyses ral stable isotopic composition, have laser-induced breakdown spectroscopy applications (14,15). emerged as powerful tracers in geosci- (LIBS), is an atomic emission tech- Despite the tremendous advances in ences, archaeology, nanotechnology, nique. Recently, these two fundamen- LA-ICP-MS and features such as high and environmental health studies. tally different techniques have been sensitivity and resolution, certain ele- Mass differences in stable isotopes also incorporated into a single commercial ments and isotopes remain largely un- give rise to fractionation during phys- instrument and the resulting LIBS–LA- measurable by LA-ICP-MS. Recurring ical processes as a result of differences ICP-MS setup has the benefits of both limiting factors include isobaric inter- in the velocities of isotopic molecules techniques. All three forms of ICP-MS, ferences, abundance sensitivity, and of the same compounds. Information namely, quadrupole, time-of-flight, detector saturation that forces masking about the differences in these isotopes and magnetic sector instruments have of specific element masses and reduced can help researchers understand phys- been used for this kind of technique sensitivity as a result of insufficient icochemical processes such as mass hyphenation. LIBS uses the character- ionization of high ionization poten- transfer and temperature changes. For istic photons generated during the re- tial elements in the argon ICP plasma. example, magnesium is one of the most laxation of the excited atomic and ionic These parameters collectively make the abundant elements on the Earth and has

magentablackcyanyellow ES1048070_LCGCSUPP0518_010.pgs 05.01.2018 15:31 ADV chromatographyonline.com May 2018 Current Trends in Mass Spectrometry 11

geochemical and biogeochemical processes. High-precision Mg isotope ratios allow us to understand geochemical pro- (a) (b) 108

107 cesses in low-Mg rocks as well as many 4 6x10 Si (I) 106 high-Mg rocks (18). Cu isotopic com- 105 position in the human body can help to 4x104 Ca (II) 104 identify the sources of Cu in the body 103 AI (I) Ca (II)

4 as well as processes that are responsible 2x10 102 AI (I) LIBS intensity (a.u.) ICP-MS signal (counts) Mn (II) Fe (I) Cu (I) 1

Zn (I) 10 for the movement of Cu in the body and Mn (II) Cu (I) Mg (I) 0 100 to understand how reactions involving 285 290 295 300 305 310 315 320 325 330 0 25 50 75 100 125 150 175 200 225 250 Mass (amu) Wavelength (nm) copper may be affected by disease. Until (c) (d) recently, measurement of these stable 2.5x104 0.10 Ca 315.9 nm 5.0x105 0.10 44Ca isotopic compositions has been carried 2.0x106 0.20 4.0x105 0.20

6 out by thermal-ionization mass spec- 5 1.5x10 0.30 3.0x10 0.30

4 2.0x105 1.0x10 trometry (TIMS), and multicollector 0.40 0.40 1.0x105 5.0x105 0.50 0.50 (MC)-ICP-MS (19). The current chal- 0.0 0.0 0.60 0.60 lenges in the measurement of nontradi-

0.70 0.70 tional stable isotopes in complex samples

0.80 0.80 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.10 0.20 0.30 0.40 0.50 0.60 0.70 by MC-ICP-MS lie in the identification and monitoring of spectral interferences, because certain molecular species can Figure 3: Representative LIBS (a) and LA-ICP-MS (b) spectra acquired from a single location of the interfere directly with the atomic ions of 2 sample. Surface (2D) distribution maps of Ca of the same 0.785 x 0.785 mm area of a bastnaesite the same nominal mass, leading to inac- rock sample obtained simultaneously by (c) LIBS and (d) LA-ICP-MS (16). curate isotope ratio determination. For example, 40Ar56Fe directly overlaps on three isotopes (24Mg, 25Mg, and 26Mg) 24Mg and 26Mg of ~8%. Mg isotopes 96Mo during molybdenum isotope ratio with relative mass difference between can be significantly fractionated during measurement, and the analysis of cer- ANALYZE EVERY MOLECULE

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Collision–reaction cell

High-energy path Low-energy path

Source Transfer optics to mass analyzer

Figure 4: Schematic diagram of a collision–reaction cell MC-ICP-MS instrument with dual path transfer optics. (Adapted with permission from Nu Instruments.)

tain isotopes such as 40Ca is not possible detectors, which dramatically shortened applications in environmental and life on traditional MC-ICP-MS instruments the analysis time down to 1–5 s for in sciences because of the introduction of because the 40Ar interference cannot be situ isotope ratio measurement over the femtosecond laser systems, which offer resolved. In addition, the low sensitiv- conventional U–Pb age determinations distinct improvements in terms of ma- ity offered in higher resolution mode is using single-collector ICP-MS instru- trix effects and elemental fractionation. another problem because for every 10- ments. Conventional multiple collec- This performance improvement is also fold increase in resolving power, there tion using the Faraday detectors can be a result of the availability of a wide va- is a concomitant decrease in sensitivity. erroneous when the signal intensities of riety of certified reference materials But recently one firm has introduced a the analytes are unstable, particularly (22). In addition, the reduction of the collision–reaction cell MC-ICP-MS in- when the laser-ablation sampling tech- internal volume of the laser-ablation strument with a novel design that incor- nique is adopted. Deterioration in both cell for faster washout time contributed porates both a “high energy” ion path the precision and accuracy of the isotope to the successful application of LA-ICP- (traditional MC-ICP-MS) and a separate ratio measurements can be a result of the TOF-MS to high-speed and high-spatial “low energy” ion path (collision–reac- slow response of the Faraday amplifier. resolution multielement analysis (23), tion cell-MC-ICP-MS) (Figure 4) to en- Daly detectors showed better long-term and also for elemental characterization able accurate and precise measurement gain stability and wide dynamic range of diverse samples such as toners, inks, of these stable isotopes to meet the re- compared to conventional electron mul- papers, gunshot residues, fragments of quirements of real applications. The in- tipliers. Other improvements include automotive paints, and samples of hair strument offers an ideal solution for the the development of mini torch that and bones in forensic studies. precise and accurate isotopic analysis of consumes only two-thirds the argon both the traditional isotope systems, and compared to a conventional ICP torch, Speciation Studies the nontraditional isotope systems. and improved spray-chamber systems Analysis of animal- and plant-based with features such as faster temperature foods for toxic elements such as Pb, Other Significant Developments equilibration and extended temperature Cd, As, Se, and Hg is of increasing im- Instrumental Improvements range (such as –25 °C to 80 °C with an portance because of raised consumer The new-generation ICP-TOF-MS in- accuracy of ±0.1 °C) for improving the awareness and the need to evaluate and struments are becoming more popular performance of ICP-MS by effectively establish regulatory guidelines for these because of high-speed, high-spatial res- improving signal stability and reducing toxic trace metals and metalloids. Com- olution, multielement and multi-isotope oxide interferences. mon examples include As, Cd, and Hg imaging that became possible as a result speciation in rice and As speciation in of advances in both LA and ICP-MS Femtosecond Lasers for LA-ICP-MS foodstuffs. For such applications, chro- technologies. Though one firm has One of the most fundamental limita- matographic techniques such as liquid developed an ICP-simultaneous mass tions of LA-ICP-MS has been a lack of chromatography (LC) and high perfor- spectrometer that uses a complemen- reproducibility, which can create prob- mance liquid chromatography (HPLC) tary metal oxide semiconductor (CMOS) lems during quantitative analysis, espe- coupled to all forms of ICP-MS continue detector to simultaneously measure all cially in heterogeneous materials where to be the methods of choice (24). masses from Li to U (20), for unknown difficulty in matrix matching between reasons, the expected success has not the sample and standard may introduce Single-Particle Analysis been achieved so far. Recently, Obayashi errors. However, LA-ICP-MS (all forms) Recently, single-particle ICP-MS and colleagues (21) have developed an with its high sensitivity, capability of iso- (spICP-MS) has become a proven ana- LA-MC-ICP-MS system equipped with tope ratio measurements, and excellent lytical technique for the characterization multiple-ion counting using three Daly spatial resolution has seen a surge in its of nanomaterials and to provide infor-

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mation pertaining to size, size distri- tion (4). Sample preparation devices with collision–reaction cell MC-ICP-MS a bution, particle number concentration, very high digestion efficiency such as better choice than TIMS. and major elemental composition with high-pressure ashers and high-pressure Though considerable advances have minimal sample perturbation (25). vessels for inorganic substances (27) and taken place in ICP-MS technology and techniques such as microwave-assisted instrumentation in recent years, chal- Quantitative Proteomics decomposing procedures for organic lenges still exist as a result of the com- Although ICP-MS does not directly substances (28) are becoming more and plex and variable matrices of different facilitate protein identification, the more popular. types of samples. All forms of ICP-MS high-temperature process inside an instrumentation including recently inductively coupled plasma leads to Quality Control emerged systems can be used for quan- complete fragmentation of all sample In all the measurements discussed here, tification only when suitable interna- molecules, leaving only their detectable there is a great need for stringent quality tional CRMs are available for every kind atomic constituents, namely metals, control measures. Because most analyt- of application. Thus, the availability of a metalloids, or heteroatoms (such as Se ical techniques, including ICP-MS, are range of CRMs, and perfect dissolution or P), which can be used as surrogates comparative, a sample of known compo- procedures, are extremely important for to detect complex molecules such as sition (reference material) is required for the success of these technologies. Novel proteins, nucleic acids, or even small accurate calibration. One of the biggest application requirements continue to organic molecules. In this emerging challenges is the availability of calibrated pop up from time to time, and bring area, ICP-MS is able to solve many chal- elemental and isotopic reference materi- challenges to instrument manufacturers. lenges in quantitative proteomics that als for every application. However, inter- are hard to address by other techniques, national organizations like the National References as clearly seen in the large number of Institute of Standards and Technology (1) R.S. Houk, V.A. Fassel, G.D. Flesch, recent publications (26). (NIST) in the United States and the H.J. Svec, A.L. Gray and C.E. Taylor, Federal Institute for Materials Research Anal. Chem. 52, 2283–2289 (1980). Miniaturization and Testing (BAM) in Germany support (2) N. Bradshaw, E.F.H. Hall, and N.E. Another emerging trend in the world accurate and compatible measurements Sanderson, J. Anal. At. Spectrom. 4, of analytical instrumentation is minia- by certifying and providing hundreds of 801–803 (1989). turization (9). Because of tremendous certified reference materials (CRMs) for (3) P. Myers, G. Li, P. Yang, and G.M. advances in microelectronics, instru- many applications. Hieftje, J. Am. Soc. Mass Spectrom. 5, ment designs, and computer technol- 1008–1016 (1994). ogy, today the majority of analytical Future Challenges (4) M. Satyanarayanan, V. Balaram, S.S. instruments have moved to the bench- and Opportunities Sawant, K.S.V. Subramanyam, V. top and, in some cases, are available in As indicated by the broad range of ex- Krishna, B. Dasaram, and C. Manik- portable or handheld options. Among amples discussed here, exciting devel- yamba, Atomic Spectrosc. 39(1), 1–15 other benefits, miniaturization can cut opments have taken place in ICP-MS in (2018). costs and make techniques more envi- recent years. ICP-MS/MS instruments (5) U. Rambabu, V. Balaram, R. Rath- ronmentally friendly by considerably further improve on the interference-re- eesh, S. Chatterjee, M.K. Babu, and reducing power and reagent require- moval capabilities of collision–reaction N.R. Munirathnam, J. Testing and ments. Although there is a consider- cells and also improve detection limits Evaluation 46(5) (2018). https://doi. able size reduction in current ICP-MS for several elements. Tandem LA-LIBS org/10.1520/JTE20160645. instruments compared to the first-gen- coupled to ICP-MS can provide unique (6) V. Balaram, Trends Anal. Chem. 80, eration instruments, no ICP-MS in- images and can map major, minor, and 83–95 (2016). strument has yet become handheld trace element and isotope distribution (7) L. Bush, Spectroscopy 30(6), 74–73 so far, but with the kind of rapid ad- information in different materials from (2015). vancements taking place currently in a single sampling event. Collision–reac- (8) C.T. Kamala, V. Balaram, M. Sa- microelectronics, chip technologies, tion cells greatly enhance the analytical tyanarayanan, A. K. Kumar, and and other allied areas, that possibility capability of MC-ICP-MS, and this an- K.S.V Subramanyam, Archives Envi- cannot be ruled out for the future. alytical tool is set to make an indelible ron. Contami. Toxicol. 68, 421–431 mark in the areas of environmental, bi- (2015). Sample Preparation Methods ological, geochemical, medical, and even (9) V. Balaram, Spectroscopy 31(10), Despite rapid developments and inno- in the emerging nanotechnology fields 40–44 (2016). vations in ICP-MS, sample preparation with its outstanding performance for the (10) T. Ohno, Y. Muramatsu, Y. Shikam- remains the main bottleneck of elemen- interference-free measurement of some ori, C. Toyama, N. Okabe, and H. tal and isotope analysis. Although laser of the most challenging, nontraditional Matsuzaki, J. Anal. At. Spectrom. 28, sampling has made it possible to conduct stable isotope systems, and thus can pro- 1283–1287 (2013). in situ analysis on solid samples directly, vide a wealth of information. Procedural most applications still require acid diges- simplicity and larger throughputs make (Continued on page 38)

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The Use of MALDI Imaging Mass Spectrometry for Interdisciplinary Research: From Metabolomics to Pesticides Matrix-assisted laser desorption–ionization (MALDI) imaging mass spectrometry allows direct, in situ, label-free measurement of proteins, peptides, lipids, small-molecule drugs and their metabolites, and other chemicals in tissues. Applications range from fundamental biological research, through environmental and toxicological science, to pharmaceutical R&D. In each case, the unique information generated by MALDI imaging has made a significant contribution to understanding important factors such as molecular and metabolic mechanisms and the transport and localization of compounds or metabolites with human, animal, or plant species. MALDI imaging can also elucidate the impact of pesticides like chlordecone on human health over the long term, and provide supporting evidence for environmental legislation as well as political and economic decisions about environmental remediation and policy.

Shannon Cornett, Mike Easterling, and Charles Pineau

ver the past decade, the demand for highly sensitive tabolites, and other chemicals in tissues. Early experiments by Oand ultrafast mass spectrometry (MS) techniques has Caprioli demonstrated MALDI ion image analysis of regions of soared, driving the innovation of a diverse array of mass rat pancreas and pituitary. Localized peptides were mapped, and analyzers for use in a broad range of applications, such as drug the authors noted that in a single spectrum from a rat pituitary discovery, ‘omics studies, drug metabolism studies, and envi- print, more than 50 ions corresponding to the peptides present ronmental sciences. Mass spectrometers are now commonplace in the tissue were observed, including precursors, isoforms, and outside of traditional analytical laboratories and the emergence metabolic fragments (1). of different ionization sources—such as direct matrix-assisted More recently, a comprehensive review of the potential of laser desorption–ionization (MALDI)—has propelled many MALDI imaging for the toxicological evaluation of environ- advances, including in MS imaging. mental pollutants was published (2). The authors concluded MALDI imaging, a technique that was first highlighted in that MALDI imaging has great potential for studies involving the late 1990s by Caprioli (1) as a sensitive tool for the study the distribution and metabolism of chemicals released into of biochemical processes in mammalian tissue sections, has the environment, and to better elucidate their mechanisms made significant contributions to the field of molecular biol- of action. ogy, particularly in the study of the transport and localization A range of mass spectrometers can be integrated into a of compounds and metabolites in biological systems, including MALDI imaging system, depending on application needs. humans, animals, and plants. MALDI time-of-flight (TOF) MS, for example, offers high throughput (with lower ability to distinguish molecules of Integrating Biology and Chemistry similar molecular weight), while magnetic resonance mass MALDI imaging allows direct, in situ, label-free measurement spectrometry (MALDI-MRMS) provides high measurement of proteins, peptides, lipids, small-molecule drugs and their me- accuracy and mass resolving power.

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MALDI Imaging in Pharmaceutical complete picture. Both techniques have tract from an early work by Castellino R&D and Cancer Research challenges. QWBA is a robust method, and colleagues in which a small area of a Throughout the 2000s, MALDI imag- and the data generated are accepted by histology section is magnified, showing ing developed along two parallel tracks: regulatory bodies around the world. regions of inflammation (3). An ion map Interest in its use as a research tool con- However, QWBA requires the use of ra- (with 50-μm spatial resolution), which tinued to grow among biologists, and at dioactively labeled probes and presents a corresponds to the same region, indicates the same time, the first publications of its composite of the total radioactivity pres- that the localization of lapatinib metabo- application in pharmaceutical research ent in the body, including any combina- lite, M10, is only in the regions associated and development (R&D) were appearing. tion of parent drug, metabolites, impuri- with the inflammation. Traditional approaches in pharmaceu- ties, and degradation products. In addition, MALDI imaging is con- tical R&D for the risk assessment of safe LC–MS analysis is performed on tributing to the development of new and efficacious drugs have been based extracts from tissue homogenates. The biomarkers of disease, particularly in on measuring the parent drug in plasma, technique cannot provide any spatial the field of cancer research. Here, the both in animal models and humans. It information and, importantly, can be heterogeneous nature of cancer samples is recognized, however, that most drug misleading. For example, if an analyte means that because the spatial distribu- targets are not located in plasma, and in the tissue is highly localized, the ex- tion and the histology of the samples is determining the relevant tissue distri- traction and homogenization process preserved, MALDI imaging is well suited bution of not only the parent drug, but will act as a dilution, masking this dis- to this work. Importantly, MALDI im- also its metabolites, would provide much tribution and showing a relatively low aging has been successfully applied to greater understanding of pharmacology concentration, sometimes even below the detection of previously unknown and toxicology. the limit of detection. proteins in cancer, as well as for recent The development of current best Against this background, MALDI advances in the biomarker discovery of practice methods for quantitative whole- imaging offers potential advantages for N- and O-glycosylation of proteins. In body autoradiography (QWBA) and liq- drug development. For example, MALDI such studies, MALDI imaging has been uid chromatography coupled with mass imaging is generating important new in- used to develop tumor-specific glycan spectrometry (LC–MS) clearly move the formation about the localization of drug biomarkers; this work shows promise for emphasis to analyzing tissues rather than compounds and important metabolites the characterization of N-linked glycans plasma, but still fall short of providing a in tissue samples. Figure 1 shows an ex- in cancer tissues (4).

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Instrument Advances Drive techniques to gain insights into the effects mental and chemical toxicity. Pineau is New Frontiers in Biology of environmental toxins on reproduction, a research director at Inserm (the French Importantly, although the most well- chemical interactions and structures in National Institute of Health and Medi- known association of MALDI imaging is the food supply, and evaluating plants cal Research) and a team leader at IRSET in pharmaceutical applications, that in- engineered for bioremediation of con- (the Research Institute for Environmen- dustry represents just one sector that ad- taminated lands. tal and Occupational Health). As part of opted the technique early. At around the One group, led by Charles Pineau, his role at IRSET, Pineau is the director same time that initial work in the phar- has been using MALDI imaging and of Protim, a core facility involved in op- maceutical industry took place, several applying it to research developmental timization and validation of new tech- institutes in France were using MALDI and reproductive biology and environ- nologies in two fields of proteomics: extensive proteome characterization of complex samples, and MALDI imaging Serial tissue sections for toxicology and clinics. H&E stain Ion images Collaborations Powering Imaging Excellence Protim is involved in two collaborative Central Central vein vein European Union (EU)–funded projects: “3D MASSOMICS” and “METASPACE.” The recently completed research project “3D-MASSOMICS” was funded as part of the EU FP7 HEALTH program, Inﬂammation with the aim to enable three-dimensional Inﬂammation (3D) label-free proteomics and metabolo- 50�m 50�m mics imaging using MALDI imaging (5). Traditional 3D imaging techniques such m/z 649.14 - M10 as computed tomography (CT), magnetic resonsance imaging (MRI), and positron emission tomography (PET) cannot be used for proteomics or metabolomics discovery studies, nor for biomarker discovery and disease pathway analysis. To address this challenge, the consortium Figure 1: Correlation of histology and MALDI imaging in dog liver sections. Adapted with permission united nine partners, including Protim, from reference 3. from six countries both from industry

(c)1 (a) (b) Exact mass ﬁltering RB RBa1s1 a2s1 Sum formula Adduct Annotation Measure of spatial chaos 22 C H O [M+Na]+ Nandrolone 8 27 34 3 0.5 23 phenpropionate FDR MSM + C39H73O8P [M+Na] PA(36:2) 10 C H NO P [M+H]+ PC(33:0); PE(36:0) 35 41 82 8 0.1 12 C H N O P [M+H]+ SM(d36:1) 41 83 2 6 0 C H NO P + PC(34:1), PE(37:1) 100 200 42 82 8 [M+Na] Number of annotations C H NO P + 44 80 8 [M+H] PC(36:4) (d)1 [M+Na]+ + C44H84NO8P [M+Na] PC(36:2) 119 C H NO P [M+H]+ PE(39:1), PC(36:1) 44 86 8 0.5 [M+Na]+ + FDR C45H78NO7P [M+Na] PE(P-40:6) PE(dm40:6) + 0.1 RB C46H78NO8P [M+H] PC(38:7) a2s2 0 100 200 Number of annotations

Figure 2: The FDR-controlled molecular annotation for three MALDI FT-ICR imaging MS datasets from rat brain coronal sections (HMDB, FDR desired=0.1): (a) Venn diagrams showing numbers of molecular annotations and a list of 10 annotations from all three datasets. (b) Exemplary ion images of four annotations (see Table SD1.2 in reference 8 for detailed information about all 10 annotations), as well as FDR curves illustrating: (c) superiority of MSM as compared to individually considered exact mass filtering and measure of spatial chaos, and (d) decrease of reliability of molecular annotation while simulating deteriorated mass accuracy and resolution by taking signals with a larger m/z tolerance. Adapted with permission from reference 8.

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13 (a) (b) (c) C10-Chlordecone hydrate

310 252 295

273 Healthy tissue 140 Healthy Healthy tissue: tissue 381 CV

212 Necrotic 153 237 area Average quantity: 355�g/g

500�m 500�m 500�m m/z 506.68 0% 15% m/z 506.68 0% 15%

Figure 3: In situ absolute quantification of chlordecone in the pathological liver of a chlordecone and CCl4 treated mouse. (a) Microscopic image of a serial section stained with H&E. (b) MALDI image corresponding to chlordecone hydrate: an average absolute quantity of chlordecone of 355 μg/g was measured in the analyzed area. (c) Local absolute quantities measured in each necrotic areas are lower than those measured in the healthy tissue (381 μg/g). The uniform distribution of the internal standard (m/z 516.71) on the tissue section confirms that the differential distribution of chlordecone is not a result of ion suppression effects.

of this molecular imaging tool (6). The review identified that the reduction of acquisition time is important in 3D imaging MS development. When using modern MS with fast analyzers (such as TOF or quadrupole time-of-flight [QTOF] technologies) and high-frequency lasers of 2–5 kHz, the pixel-by-pixel method of data acquisition requiring high- acceleration high-precision positioning stage becomes a bottleneck. Advanced high-performance imaging MS was reported that uses 5–40 kHz lasers, providing an acquisition speed of 20 pixels per second. METASPACE, funded as part of the EU’s Horizon 2020 research and innovation program, is an ongoing project for Protim (7). It is a specialist platform that hosts an engine for metabolite annotation of MALDI imaging data as well as a spatial metabolite knowledge base of the metabolites from hundreds of public datasets provided by the community. The Figure 4: Molecular image of sperm transit in the rat epididymis by MALDI imaging MS. Peptides METASPACE platform is developed by specific to maturing spermatozoa during epididymal transit can be visualized simultaneously. Signal software engineers, data scientists, and overlay of three specific peptides corresponding to m/z 5470, 6177, and 18,746 is visualized in MS experts from the Alexandrov team different colors. An 80-μm lateral image resolution is mandatory to resolve the organ structures. at the European Molecular Biology Lab- oratory (EMBL). As part of this project, and research, to develop statistical meth- A review by the coordinators of the imaging MS was used for spatial metab- ods for the reproducible collection of 3D consortium, Palmer and Alexandrov, olomics analysis with the aim to create a MALDI imaging data, and for unsuper- aimed to assess and increase the knowl- bioinformatics tool for automated metab- vised and supervised statistical analysis edge of 3D imaging MS in the analytical olite annotation, in high mass resolution and interpretation of this data. community, to accelerate the innovation imaging MS (8). The development of an

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signal variations. Finally, depending on the physicochemical properties of the studied molecule and the composition of the matrix solution, the extraction of the compound of interest occurring during matrix application can vary in efficiency. Pineau’s team was able to develop a robust method for in situ quantification directly on the tissue sections, published in 2014 (12). Figure 3 shows the in situ absolute quantification method (ISAQ) of chlordecone that was demonstrated in the Figure 5: Three-dimensional mass spectrometry imaging view of a rat epididymis head. PLSA pathological liver of a chlordecone- and computation in SCiLS software with restricted mass range (m/z 250–1650). Left: m/z corresponding CCl4-treated mouse. to a metabolite localized in the vas efferens area. Right: m/z corresponding to a metabolite expressed The group successfully developed a along the tubule. method for the quantification of small molecules by MALDI imaging, based on algorithm to efficiently mine 10–100 gi- to the chemical and the soil and coastal the combination of labeled normalization gabyte datasets was established, and the waters were polluted. with the calculation of a correlation curve results of the annotation are shown in Initial work by epidemiologist Luc between MALDI imaging and GC as a Figure 2. Multigner (a team leader at the IRSET conventional quantification technique. institute), published in 2010, confirmed This method was successfully applied to Analysis of Human that chlordecone was responsible for a the quantification of chlordecone in the Exposure to Chlordecone significant increase in the risk of pros- mouse liver, and is the first example of In addition to the 3D-MASSOMICS and tate cancer, the cause of half the cancers the application of MALDI imaging to the METASPACE projects, Pineau’s team at detected on the two islands (10). quantification of a pesticide in tissues. Protim has a number of active research In parallel, the group at Protim was Although this study represents an im- projects investigating how exposure to progressing new laser technology to important step forward in the capabilities chemicals in the environment impacts prove the resolution of MALDI imag- of MALDI imaging, chlordecone has a human health. Traditionally, limits and ing. A breakthrough publication in 2011 half-life of 30 years, which means that the guidelines from the U.S. Environmen- showed for the first time that it was possi- environment in Guadaloupe is predicted tal Protection Agency (EPA) and other ble to acquire MALDI images of proteins to be polluted for more than 500 years official bodies regulate acceptable envi- in the 10 kDa range at 20-μm lateral res- to come. Scientists continue to monitor ronmental levels of toxic chemicals. De- olution on a commercial instrument (11). various population groups on the islands pending on the toxin type and matrix Rat testis was used as a relevant model to to assess the ongoing impact on male fer- (air, water, or soil), compliance with these demonstrate that this improved resolu- tility and child development and behav- limits is typically measured using dedi- tion corresponded to complex anatomical ior. For example, studies of the effects of cated environmental chemistry meth- features at the cellular level. The correla- chlordecone exposure on pregnancy du- ods (ECMs) such as gas chromatography tion of molecular signals was successfully ration and the risk of preterm birth found (GC) and LC, often in tandem with elec- established with the development of germ that maternal exposure to chlordecone tron-capture detection (ECD) methods (a cells within the seminiferous epithelium. was strongly associated with a shorter full list of techniques and ECM methods This level of sensitivity was deemed pregnancy duration and an increased can be found in reference 9). However, essential to the next stage of the chlor- risk of preterm birth, regardless of the those measurements do not shed light decone research and the work to investi- method of onset of labor (spontaneous on the mechanisms and potential impact gate the localization of chlordecone in the or induced). The hormonal, estrogenic, on human health that these substances in body and quantify it directly on the tissue and progestogenic properties of chlorde- the environment might have. sample. The development of MALDI im- cone are thought to be the cause of this Several IRSET teams in Rennes, aging for toxicological evaluation of small association (13). France, are working on analysis of the molecules requires solid quantification pesticide chlordecone, which was used methods. However, such quantification Using 3D Imaging to Advance in Martinique and Guadeloupe to com- is complicated with MALDI imaging for Knowledge of Spermatogenesis bat banana weevil until 1993. Chlorde- several reasons. First, endogenous spe- In addition to the work on the impact of cone is a known endocrine disrupter and cies present in a tissue section can affect chlordecone and other toxic compounds carcinogen, and its use has been banned the intensity of the ion of interest and (bisphenol A and glyphosate, for exam- worldwide since 1990. Before the ban, a can lead to varying ionization efficiency ple), Pineau and the Protim team are con- significant proportion of the population across the section. Second, the heteroge- tributing to wider applications of MALDI in the French West Indies was exposed neity of matrix crystallization can induce imaging in human reproductive biology.

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With more than 30 years of experience, the localized distribution of peptides, (3) S. Castellino, M.R. Groseclose, and D. Pineau is a renowned specialist in sper- proteins, and small molecules in tis- Wagner, Bioanalysis 3(21), 2427–2441 matogenesis. He has made significant sues. Applications range from funda- (2011). contributions to the development of pro- mental biological research, through en- (4) M.J. Kailemia, G. Xu, M. Wong, Q. Li, E. teomics and integrative proteomics tech- vironmental and toxicological science, Goonatilleke, F. Leon, and C.B. Lebrilla, nologies and their application to answer to pharmaceutical R&D. In each case, Anal. Chem. 90, 208–224 (2018) biological and clinically relevant prob- the unique information generated by (5) 3D-MASSOMICS, University of Bremen, lems in the field of testicular pathophysi- MALDI imaging has made a signifi- http://eu3dmassomics.uni-bremen.de. ology and reproductive toxicology. Now, cant contribution to understanding im- (6) A.D. Palme and T. Alexandrov, Anal. MALDI imaging is playing a key role in portant factors such as molecular and Chem. 87, 4055–4062 (2015). his further investigations of the complex metabolic mechanisms and the trans- (7) METASPACE http://metaspace2020. process of sperm maturation. port and localization of compounds eu/#/about. The epididymis is a small organ lo- or metabolites with human, animal, (8) A. Palmer, P. Phapale, I. Chernyavsky, R. cated next to the testicle. Immature or plant species. As highlighted in this Lavigne, D. Fay, A. Tarasov, V. Kovalev, spermatozoa are formed in the testis article, MALDI imaging can also show J. Fuchser, S. Nikolenko, C. Pineau, and are morphologically complete, how pesticides like chlordecone impact M. Becker, and T. Alexandrov, Nature but lack functionality. Gametes transit human health over the long term, and Methods 14, 57–60 (2017). through the epididymis, acquiring mo- provides supporting evidence for envi- (9) Environmental Chemistry Methods tility and the ability to fertilize during ronmental legislation as well as polit- (ECM), Analytical Methods and Pro- this maturation process. Nuclear gene ical and economic decisions about re- cedures for Pesticides, (U.S. Environ- transcription is switched off at the mediation and environmental policy. mental Protection Agency, Washington, sperm maturation level, and the process Arguably still in its infancy, continu- D.C.) https://www.epa.gov/pesti- is driven by an array of sequential pro- ous improvements in MALDI imaging cide-analytical-methods/environmen- tein modifications that are inextricably systems have made the technique more tal-chemistry-methods-ecm-index-0-9. linked to the complex epididymal lumi- accessible, more reliable, and easier (10) L. Multigner, J.R. Ndong, A. Giusti, M. nal microenvironment. When a sper- to use, and data analysis has become Romana, H. Delacroix-Maillard, S. Cord- matozoon passes from caput to cauda, both more sophisticated and more au- ier, B. Jégou, J.P. Thome, and P. Blan- numerous biochemical events occur in tomated. MALDI imaging looks set to chet, J. Clin. Oncol. 28(21), 3457–3462 its subdomains, including post-trans- play an increasingly important role in (2010). lational modifications, proteolytic the coming years. (11) M. Lagarrigue, M. Becker, R. Lavigne, processing, protein redistribution and S-O Deininger, A. Walch, F. Aubry, D. disappearance, and integration of new Acknowledgments Suckau, and C. Pineau, Mol. Cell. Pro- components, thus leading to a dynamic This work was supported in part by teomics 10(3), M110.005991 (2011). remodeling of the sperm surface. Charles Pineau, PhD, the research di- (12) M. Lagarrigue, R. Lavigne, E. Tabet, The unique ability of MALDI imaging rector of Inserm and the director of the V. Genet, J.P. Thomé, K. Rondel, B. to visualize molecules in situ has signifi- Protim Core Facility; Richard M. Cap- Guével, L. Multigner, M. Samson, and cantly advanced the study of this complex rioli, a professor in the Departments C. Pineau, Anal. Chem. 86, 5775−5783 biology. Figure 4 shows a two-dimensional of Biochemistry, Chemistry, Phar- (2014). (2D) image of a rat epididymis head with macology, and Medicine at Vander- (13) P. Kadhel, C Monfort, N. Costet, F. highlighted peptides possibly involved in bilt University School of Medicine, in Rouget, J.P. Thomé, L. Multigner, and the maturation of sperm during its tran- Nashville, Tennessee; Hélène Rogniaux, S. Cordier, Am. J. Epidemiol. 179(5), sit through the organ (14). Figure 5 illus- PhD, a research engineer at the Bio- 536–544 (2014). trates the latest work in Pineau’s lab: the polymères, Interactions, Assemblages (14) Protim, https://www.protim.eu/index. creation of 3D images (15). The process of (BIA) unit of the French National Insti- php/en/. capturing a 3D MALDI imaging model of tute for Agricultural Research (INRA); (15) R. Lavigne et al., in preparation. an organ begins with making many hun- and Dimitri Heintz, PhD, the head of dreds (or thousands) of sections through the Platform Imaging Mass Spectrom- Shannon Cornett, PhD, is the Imaging the organ. Each section is measured and etry PIMS Metabolomics laboratory Business Manager at Bruker Daltonics in the data are combined in software to cre- (IBMP) in Strasbourg, France. Nashville, Tennessee. Mike Easterling, ate the final picture. The resulting data sets PhD, is the Imaging Mass Spectrometry are huge—in the terabyte range—but once References Manager at Bruker Daltonics in Los completed, a dataset can be mined for in- (1) R.M. Caprioli, T.B. Farmer, and J. Gile, Angeles, California. Charles Pineau, PhD, formation for many years. Anal. Chem. 69(23), 4751–4760 is a Research Director at Inserm and the (1997). Director of the Protim Core Facility at the Summary (2) M. Lagarrigue, R.M. Caprioli, and C. University of Rennes 1, in Rennes, France. MALDI imaging has emerged as a tech- Pineau, J. Proteomics 144, 133–139 Direct correspondence about this article to nique that offers deep understanding of (2016). [email protected] ◾

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Resolving Volume-Restricted Metabolomics Using Sheathless Capillary Electrophoresis– Mass Spectrometry The analytical toolbox used in present-day metabolomics encounters difficulties for the analysis of limited amounts of biological samples. Therefore, a significant number of crucial biomedical and clinical questions cannot be addressed by the current metabolomics approach. Capillary electrophoresis–mass spectrometry (CE–MS) has shown considerable potential for the profiling of polar and charged metabolites in volume-restricted or mass-limited biological samples. This article considers advances that significantly improved the performance of CE–MS for in-depth metabolic profiling of limited sample amounts. Attention is also devoted to various technical aspects that still need to be addressed to make CE–MS a viable approach for volume-restricted metabolomics. Rawi Ramautar major challenge in bioanalysis and metabolomics is the tabolites in ultrasmall biological samples, as has been recently Aprofiling of (endogenous) metabolites in volume-re- demonstrated for the analysis of CSF from mice and extracts from stricted and often scarce biological samples. Though the small tissues or a single cell (4–7). Moreover, CE (denoting here analytical techniques currently used in metabolomics are power- capillary zone electrophoresis [CZE]) separates compounds based ful, the need for relatively large amounts of sample for pre-analyt- on differences in their intrinsic electrophoretic mobility, which ics and injection prevent their use in many biomedical and clinical is dependent on the charge and size of the analyte and, therefore, applications. For example, metabolic profiling of small-volume well-suited for the analysis of polar and charged metabolites. As biological samples, such as cerebrospinal fluid (CSF) from mouse the separation mechanism of CE is fundamentally different from models, spheroids or microtissues, liquid biopsies, and samples chromatographic-based separation techniques, a complementary from microfluidic organ-on-a-chip systems, is seriously hindered view on the composition of (endogenous) metabolites present in by the current analytical technologies because of the limited sam- a given biological sample is provided. In comparison with chro- ple material. The standard analytical techniques also do not allow matographic-based methods the separation efficiency of CE is a maximum amount of biochemical information from valuable very high because there is no mass transfer between phases, and and scarcely available biological samples to be obtained because under perfect experimental conditions the only source of band the material is completely consumed for a single metabolomics broadening in CE is from longitudinal diffusion. measurement. Despite important developments in analytical sep- Over the past few years, various new CE–MS approaches arations technology over recent years, the analysis of small-vol- have been developed that show a strong potential for improv- ume biological samples remains a challenging task. Therefore, ing the sensitivity and metabolic coverage in metabolomics (8). there is a critical need for the introduction of analytical methods This article focuses on advances that significantly improved to allow highly sensitive metabolic profiling of volume-restricted the analytical performance—especially with regards to im- or mass-limited biological samples. proving the metabolic coverage—of CE–MS for volume-re- Capillary electrophoresis–mass spectrometry (CE–MS) can stricted metabolomics studies. be considered an attractive microscale analytical method for metabolomics because in CE nanoliter injection volumes are Sheathless CE–MS Using a Porous Tip Sprayer used from microliter sample amounts or less in the injection vial CE is essentially a low-flow microscale separation technique (1–3). Therefore, CE–MS is highly suited for the profiling of me- that reaches its optimal separation performance at very low flow

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tion and an improved transfer of ions to the MS system (9–11). Moreover, at very x106 low flow rates (≤20 nL/min) ion suppres- 5 sion is significantly reduced, resulting in an improved concentration sensitivity (9), 4 which is important for ultratrace detection of metabolites in limited sample amounts. 3 In a conventional CE system, both ends of the separation capillary are immersed 2 in buffer vials to which electrodes are Intensity (counts) added to provide a high voltage gradient. 1 To couple CE to MS, the outlet vial must be replaced by an interface to close the 0 electrical circuit and to provide contact 5 10 15 20 25 with the ESI stream. Therefore, a CE–MS Migration time (min) interface needs to apply voltage to the capillary outlet while maintaining inde- Figure 1: Metabolic profile (m/z 65–1000) of mouse cerebrospinal fluid obtained with sheathless CE– pendent CE and ESI electrical circuits. A MS using a porous tip sprayer. Conditions: separation buffer: 10% acetic acid (pH 2.2); electrophoretic sheath-liquid interface and various other separation at +30 kV; sample injection: circa 45 nL. Adapted with permission from reference 4. interfacing techniques have been developed to allow the coupling of CE to MS. rates—typically in the range of 20 nL/ is, under (near-) zero electroosmotic flow Until now, most CE–MS-based metabo- min to 100 nL/min—depending on the conditions. The inherently low flow rates lomics studies have been performed with pH of the separation buffer when using of CE are also useful with regards to the a sheath-liquid interface (12). CE–MS ap- a bare fused-silica capillary. A high sep- electrospray ionization (ESI) mechanism. proaches using a sheath-liquid interface for aration resolution is obtained in CE by In ESI, smaller droplets are generated metabolomics were first developed by Soga merely separating the compounds based under low flow separation conditions, and coworkers (13,14). The sheath-liquid on their electrophoretic mobilities—that which results in a more efficient desolva- interface, originally designed by Smith and

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valuable and precious mouse CSF sample, enabling repeatability studies and, even m/z 284,1157 9e4 m/z 76,0462 more interesting, analysis of the same m/z 118,0943 m/z 130,0945 sample at different separation conditions 8e4 m/z 192,1707 m/z 90,0624 m/z 133,0693 to further increase the metabolic coverage. 7e4 m/z 120,0888 m/z 120,0735 Performing multiple analyses on a single, m/z 106,0575 6e4 m/z 134,0532 scarce biological sample is not possible m/z 150,0674 m/z 116,0786 5e4 with conventional analytical techniques. m/z 156,0862 m/z 132,1103 Apart from volume-restricted biologi- 4e4 m/z 147,1219 m/z 130,0582 m/z 147,0856 cal samples, such as mouse CSF, there is

Intensity (counts) 3e4 m/z 182,0921 m/z 182,0588 currently a strong interest in analytical m/z 166,0963 2e4 m/z 241,0558 tools capable of providing highly sensitive m/z 137,0543 m/z 175,1296 metabolic profiles for microscale cell cul- 1e4 m/z 132,0852 m/z 537,9050 m/z 148,0695 ture samples. The performance of sheath- 0e0 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 less CE–MS has therefore been assessed Time (min) for the profiling of cationic metabolites in an extract from approximately 10,000 Figure 2: Multiple extraction ion electropherograms for a selected number of metabolites obtained HepG2 cells. Figure 2 shows that a rea- with sheathless CE–MS using a porous tip sprayer in an extract of HepG2 cells. Conditions: separation sonable number of metabolites could be buffer: 10% acetic acid (pH 2.2); electrophoretic separation at +30 kV; sample injection: circa 60 nL. detected by sheathless CE–MS using an coworkers (15), has been used for a broad transport of ions and electrons through injection volume of 60 nL, which in this range of bioanalytical applications with ac- the capillary wall while spraying the CE case equals the aliquot of only 5 HepG2 ceptable analytical figures of merit. How- effluent into the nano-ESI-MS system. cells. An improvement of the injection ever, the sheath liquid is generally provided The sheathless porous tip design is espe- part is needed to further enhance the de- at a flow rate between 2 μL/min to 10 μL/ cially useful for interfacing narrow (<30 tection sensitivity of the sheathless CE–MS min, thereby significantly diluting the CE μm internal diameter [i.d.]) capillaries method; however, the results obtained so effluent and resulting in compromised de- and for low flow-rate (<20–30 nL/min) far clearly indicate the strong potential of tection sensitivities for metabolomics ap- nano-ESI-MS analyses (26). the method for metabolic profiling of lim- plications. Moreover, this flow rate is not ited sample amounts. compatible with nano-ESI-MS. However, In-Depth Metabolic Profiling of Recently, the utility of the sheathless an important benefit of the sheath-liquid Volume-Restricted Samples CE–MS method was also examined for interface is that the composition can be The capabilities of CE–MS using a sheath- the profiling of anionic metabolites in modified to improve the ionization effi- less porous tip interface for global pro- biological samples (28), using the same ciency without affecting the selectivity and filing of cationic metabolites in human separation conditions used for the pro- efficiency of the electrophoretic separation urine have been assessed (27). Low nano- filing of cationic metabolites—only the (16,17). The influence of these agents on molar limits of detection (LODs) were MS detection and CE separation voltage metabolomics studies by CE–MS still obtained for a wide range of metabolite polarity were switched. A wide range needs to be explored. Overall, as both CE classes in human urine by using only an of anionic metabolite classes could be and ESI-MS perform optimally at low injection volume of 9 nL. As a follow-up profiled under these conditions, includ- flow-rate conditions, the coupling of CE to this work, the potential of sheathless ing sugar phosphates, nucleotides, and to MS should preferably be performed via CE–MS for metabolic profiling of very organic acids. An injection volume of an interface that effectively makes use of minute amounts of biological samples 20 nL resulted in nanomolar detection the intrinsically low flow separation prop- was assessed. To this end, mouse CSF limits, which corresponded to a signif- erty of CE and the improved ESI efficiency was selected as a typical example of a bi- icant enhancement when compared to under these conditions. ological material only available in minute the micromolar detection limits typi- At present, the development of new or quantities, that is, only a few microliters cally obtained with classical sheath-liquid improved interfacing designs for CE–MS can be obtained under proper conditions CE–MS methods. Structural isomers of and the evaluation of their potential for (4). Figure 1 shows a metabolic profile ob- phosphorylated sugars as well as isobaric bioanalysis and metabolomics remains an tained by sheathless CE–MS for mouse metabolites could be selectively ana- active area of research (18–25). So far, the CSF after only a 1:1 dilution with water, lyzed by the proposed sheathless CE–MS most encouraging results for volume-re- thereby fully retaining sample integrity. By method without using any derivatization. stricted metabolomics studies have been using an injection volume of 45 nL from a The approach was used for the profiling obtained by CE–MS using a sheathless vial containing only 2 μL of a 1:1 diluted of anionic metabolites in extracts from porous tip interface. In this design, which CSF, more than 300 compounds could the glioblastoma cell line. Around 100 was developed by Moini (19), a porous be observed. As only 45 nL of the sample compounds were found for an injection capillary tip inside a cylindrical metal was consumed, the proposed method al- volume of 20 nL, corresponding to the tube maintains electrical contact via lows multiple analyses on a single highly content of approximately 400 cells.

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Future Directions grant scheme of the Netherlands Orga- (17) G. Bonvin, S. Rudaz, and J. Schappler, The examples highlighted here clearly indi- nization for Scientific Research (NWO Anal. Chim. Acta 813, 97–105 (2014). cate the ability of sheathless CE–MS using Veni 722.013.008). (18) E.J. Maxwell, X. Zhong, H. Zhang, N. van a porous tip sprayer for in-depth metabolic Zeijl, and D.D. Chen, Electrophoresis 31, profiling of minute amounts of sample Conflict of Interest 1130–1137 (2010). material. Although (sub-)nanomolar de- The author has no other relevant affilia- (19) M. Moini, Anal. Chem. 79, 4241–4246 tection limits can be obtained for a wide tions or financial involvement with any (2007). range of polar compounds by only using organization or entity with a financial (20) R. Wojcik, O.O. Dada, M. Sadilek, and N.J. nanoliter injection volumes, as required for interest in or financial conflict with the Dovichi, Rapid Commun. Mass Spec- trace-level analysis of metabolites in lim- subject matter or materials discussed in trom. 24, 2554–2560 (2010). ited sample amounts, the next important the manuscript apart from those disclosed. (21) X. Guo, T.L. Fillmore, Y. Gao, and K. Tang, step is to examine the utility of sheathless Anal. Chem. 88, 4418–4425 (2016). CE–MS for large-scale volume-restricted References (22) S.B. Choi, M. Zamarbide, M.C. Manzini, metabolomics studies. Hirayama and co- (1) W. Zhang, T. Hankemeier, and R. Ram- and P. Nemes, J. Am. Soc. Mass Spec- workers have recently shown that a single autar, Curr. Opin. Biotechnol. 43, 1–7 trom. 28, 597–607 (2017). sheathless porous tip capillary can be used (2017). (23) T.T. Nguyen, N.J. Petersen, and K.D. Rand, for more than 180 successive runs of a 10- (2) N.L. Kuehnbaum and P. Britz-McKibbin, Anal. Chim. Acta 936, 157–167 (2016). fold diluted human urine sample (29). Still, Chem. Rev. 113, 2437–2468 (2013). (24) V. Gonzalez-Ruiz, S. Codesido, J. Far, S. the long-term performance of sheathless (3) A. Slampova and P. Kuban, J. Chro- Rudaz, and J. Schappler, Electrophoresis CE–MS for volume-restricted metabolom- matogr. A 1497, 164–171 (2017). 37, 936–946 (2016). ics needs to be assessed in more extended (4) R. Ramautar et al., Anal. Bioanal. Chem. (25) J. Krenkova, K. Kleparnik, J. Grym, J. studies analyzing large numbers of diverse 404, 2895–2900 (2012). Luksch, and F. Foret, Electrophoresis 37, biological samples. (5) J.X. Liu, J.T. Aerts, S.S. Rubakhin, X.X. 414–417 (2016). When using a highly sensitive mi- Zhang, and J.V. Sweedler, Analyst 139, (26) J.M. Busnel et al., Anal. Chem. 82, 9476– croscale analytical method for in-depth 5835–5842 (2014). 9483 (2010). metabolic profiling, attention should (6) R.M. Onjiko, S.A. Moody, and P. Nemes, (27) R. Ramautar, J.M. Busnel, A.M. Deelder, also be paid to the design of reliable Proc. Natl. Acad. Sci. USA 112, 6545– and O.A. Mayboroda, Anal. Chem. 84, sampling techniques for volume-re- 6550 (2015). 885–892 (2012). stricted or mass-limited samples. Nemes (7) R.M. Onjiko, E.P. Portero, S.A. Moody, (28) C. Gulersonmez, S. Lock, T. Hankemeier, and coworkers have recently developed a and P. Nemes, Anal Chem., in press and R. Ramautar, Electrophoresis 37, microprobe single-cell CE–MS method, (2017). 1007–1014 (2016). which integrates capillary microsampling, (8) P.W. Lindenburg, R. Haselberg, G. Rozing, (29) A. Hirayama, M. Tomita, and T. Soga, An- microscale metabolite extraction, and CE– and R. Ramautar, Chromatographia 78, alyst 137, 5026–5033 (2012). MS analysis, for in situ mass spectrometric 367–377 (2015). (30) A. Gjelstad and K.F. Seip, Bioanalysis 7, profiling of metabolites in single cells (7). (9) A. Schmidt, M. Karas, and T. Dulcks, J. 2133–2134 (2015). It is anticipated that microprobe single-cell Am. Soc. Mass Spectrom. 14, 492–500 (31) R.J. Raterink, P.W. Lindenburg, R.J. CE–MS will allow metabolomics studies (2003). Vreeken, and T. Hankemeier, Anal. in larger populations of single cells and (10) M. Wilm and M. Mann, Anal. Chem. 68, Chem. 85, 7762–7768 (2013). other model organisms. Proper sample 1–8 (1996). pretreatment strategies are needed for the (11) G.A. Valaskovic, N.L. Kelleher, and F.W. Rawi Ramautar studied both phar- selective extraction of polar and charged McLafferty, Science 273, 1199 –1202 macochemistry and analytical sciences at metabolites from minute amounts of sam- (1996). the Vrije Universiteit of Amsterdam, The ple. In this context, it would be interesting (12) A. Hirayama, M. Wakayama, and T. Soga, Netherlands. In 2010, he completed his to evaluate strategies like in-line or on-line TrAC, Trends Anal. Chem. 61, 215–222 PhD on the development of capillary elec- solid-phase extraction (SPE)-CE–MS or to (2014). trophoresis–mass spectrometry methods explore the possibilities of electrodriven (13) T. Soga, Y. Ohashi, Y. Ueno, H. Naraoka, for metabolomics at Utrecht University, sample pretreatment techniques, such M. Tomita, and T. Nishioka, J. Proteome The Netherlands. Intrigued by metabolo- as electromembrane extraction or three- Res. 2, 488–494 (2003). mics for disease prediction and diagnosis, phase electroextraction (30,31). Overall, (14) T. Soga, Y. Ueno, H. Naraoka, Y. Ohashi, Rawi switched to the Leiden University further developments in this area may M. Tomita, and T. Nishioka, Anal. Chem. Medical Center, The Netherlands, to result in a more viable CE–MS approach 74, 2233–2239 (2002). broaden his horizon on this topic. for probing the polar metabolome in vol- (15) R.D. Smith, C.J. Barinaga, and H.R. Currently, he is a principal investigator ume-restricted samples. Udseth, Anal. Chem. 60, 1948–1952 (tenured) at the Leiden Academic Center (1988). for Drug Research of the Leiden University Acknowledgments (16) T.J. Causon, L. Maringer, W. Buchberger, where his group is developing microscale Rawi Ramautar would like to acknowl- and C.W. Klampfl, J. Chromatogr. A 1343, analytical workflows for volume-restricted edge the financial support of the Veni 182–187 (2014). biomedical problems. ◾

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Liquid Chromatography– Atmospheric Pressure Photoionization-Mass Spectrometry Analysis of the Nonvolatile Precursors of Rancid Smell in Mayonnaise

Monitoring lipid oxidation during the shelf life of lipid-containing food emulsions, such as mayonnaise, is challenging. It is, however, essential for the development of improved, consumer-preferred products. Determining the nonvolatile lipid oxidation products (NONVOLLOPS), the precursor compounds for rancidity, is required to determine the effectiveness of product stabilization technologies. A method based on normal-phase liquid chromatography with atmospheric pressure photoionization-mass spectrometry (LC–APPI-MS) was developed for this purpose. The inclusion of a size-exclusion chromatography (SEC) step was needed to remove interfering diacylglycerides and free fatty acids from the samples. The combined SEC and normal-phase LC–APPI-MS method allowed the identification of a wide range of oxidized species including hydroperoxides, oxo-2½ glycerides, epoxides, and other oxidized species. The method was found to be more suitable for the analysis of large sample sets. The relative levels of NONVOLLOPS from both ambient and accelerated stability tests could be determined. The results were compared to hexanal measurements. The data showed that NONVOLLOPS predict the rancidity of different formulations in a much earlier stage during shelf-life tests, providing valuable information for future product development.

Boudewijn Hollebrands and Hans-Gerd Janssen

he taste, flavor, and texture of lipid-rich foods, such can undergo oxidation and hydrolysis reactions during the Tas mayonnaises and dressings, strongly depend on product’s shelf life, forming volatile and nonvolatile lipid the composition of the oil used and the emulsification oxidation products. Even at very low levels the volatile lipid methods applied during the preparation of the product. oxidation products can cause the typical rancid off-smell of Making consumer-preferred products with healthier, un- aged oil. High amounts of nonvolatile lipid oxidation prod- saturated edible oils is a key challenge for the food industry. ucts could even adversely affect the consumer’s health (1,2). The unsaturated fatty acids in the triacylglycerols (TAGs) Being able to accurately monitor lipid oxidation products

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is therefore crucial for safeguarding products of the oxidation reaction, but work that normal-phase high perfor- consumer safety and to study novel, are actually unstable intermediates. The mance liquid chromatography (HPLC) natural stabilization technologies that analytical protocols applied for sample with electrospray MS detection gives suppress lipid oxidation reactions in preparation and analysis should hence detailed information on the nonvola- lipid-rich foods. be very mild—and even then decompo- tile lipid oxidation products present in A clear drawback of the healthier sition of labile intermediates might still oxidized oils (9). The method performs unsaturated fatty acids oleic, linoleic, occur. A second parameter to consider is a polarity-based class-separation of the and linolenic acid is their much higher the complexity of the mixtures obtained. oxidized TAGs, providing information amenability for oxidation reactions. Different classes of oxidized species are on groups such as the hydroxyperoxy-, The processes occurring during lipid present, each consisting of a wide range hydroxyl-, epoxy-, or oxo (ketone or oxidation so far are not fully under- of positional isomers and covering the aldehyde)-TAGs, or TAGs with com- stood, but it is believed that most reac- entire chain length distribution. binations of these chemical function- tions proceed through radical initiation We have demonstrated in previous alities. This method remains challeng- (3). First, peroxidized and hydroperoxi- dized TAGs are formed that are precursors for the later volatiles. Dissociation reactions can then result in the loss of CYLINDER STORAGE DRIVING YOU MAD? volatile compounds, such as aldehydes and ketones, and the formation of so- called “2½ glycerides.” Other reaction pathways can lead to the formation of numerous nonvolatile oxidation products, such as hydroxy-TAG epoxides, TAGs with aldehyde functionalities, and polymers. Understanding the formation of these precursors is crucial for developing new stabilization technologies. The broad range of TAGs in edible oils combined with the wide variety of possible oxidation reactions makes the analysis of the nonvolatile precursors of the rancid smell very complex. A number of fast and simple meth- 421 10 ods have been applied to monitor oxi- 24 dation in oils. These include measuring the peroxide value (4,5) or the 2-thio- ASMS Booth barituric acid value (6) of the oil. Al- ACS–Fall Booth though important and widely used, these measurements are highly em- pirical and unspecific (7). In addition, BRING NEW LIFE TO YOUR PRODUCTIVITY they do not really provide information WITH H , N AND ZERO AIR ON-DEMAND. down to the molecular level, which is 2 2 needed to build a true understanding of the complex interplay of oxidation • Consistent Purity reactions. Chromatography combined • Consistent Pressure with mass spectrometric detection is • Proven Safe the method of choice for sensitive and selective analysis of both the volatile • Cost Effective and nonvolatile intermediate and final • Eliminates Cylinder Storage lipid oxidation products. Volatile components are commonly measured by gas and Delivery Issues chromatography–mass spectrometry (GC–MS) headspace analysis (8). In the Visit us on-line or call for a consult chromatographic analysis of nonvolatile with one of our sales engineers today. oxidation products two factors must be considered. First, it should be realized that many of these products are not end +1-203-949-8697 www.ProtonOnSite.com magentablackcyanyellow ES1048053_LCGCSUPP0518_025.pgs 05.01.2018 15:22 ADV 26 Current Trends in Mass Spectrometry May 2018 chromatographyonline.com

ing, primarily because of limitations in quires the samples to be free of water. solvents, but it is time consuming. Tak- the repeatability and reproducibility. Food products, such as mayonnaise and ing this all together, the normal-phase Sensitive electrospray ionization of the spreads, however, are emulsions con- LC–electrospray MS method and the nonpolar compounds requires the use taining up to 75% water. The products sample preparation required are far of a post-column additive to facilitate from the lipid oxidation reaction can from ideal for studying the initial stages ionization, which unfortunately rap- accumulate in either the oil phase, the of lipid oxidation, especially if large idly pollutes the ion source. Another water phase, or at the oil–water inter- sample sets have to be analyzed. limitation is the sample preparation. face. Freeze-drying effectively removes In this article, we describe a novel Normal-phase chromatography re- water, allowing extraction with organic analytical approach for determining the nonvolatile oxidation products (NONVOLLOPS) in mayonnaise samples from (accelerated) shelf-life tests. Epoxy–Oxo 2½ -glycerides OOH Highly oxidized The method uses atmospheric pressure photoionization (APPI) MS. The interpretation of the mass spectra is discussed and methods to distinguish MS fragmentation from overlap of nonre- acted species are evaluated. Eventually, the nonvolatile precursors of the off- smell compounds of aged mayonnaise

Abundance (%) are determined. Understanding off- smell formation is built by correlating the results of the new NONVOLLOPS methods with those of classical hexanal measurements. 10 15 20 25 30 35 40 45 50 55 60 65 Retention time (min) Experimental Materials Figure 1: Lipid oxidation products in oil extracted from aged mayonnaise. Stable isotope labeled cholesterol D6

4 +APPI Scan (rt: 40.171 min) x10 603.4 6.0 879.6 4.0 475.4 647.4 419.3 2.0 281.2 663.4 903.7 530.4 855.6 263.1 339.2 397.3 445.4 489.4 577.5 621.4 687.5 0 5 +APPI Scan (rt: 44.461 min) x10 1.6 897.6

1.2 879.6

0.8 869.6 617.5 0.4 475.4 589.5 797.6 419.3 493.4 533.4 647.4 827.6 957.7 MS Response 0 5 +APPI Scan (rt: 47.052 min) x10 893.6 2.0 1.5 877.6 1.0 493.3 753.5 475.4 0.5 419.3 603.5 743.5 771.5 853.6 339.3 397.4 577.4 647.4 0 300 400 500 600 700 800 900 1000 Mass-to-Charge ( m/z)

Figure 2: Mass spectra of multiple coeluted lipid oxidation products and neutral lipids.

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at a flow rate of 0.3 mL/min using a linear gradient of n-hexane (Solvent A) 280 and 9:1 (v/v) n-hexane–IPA (solvent B) TAG programmed as follows: 5 min at 3% B, in 25 min to 8% B, in 10 min to 25%

200 B, 10 min at 25% B, in 2 min to 50% B, 5 min at 50% B, in 1 min to 3% B, and finally 32 min at 3% B. The total

120 run time was 90 min. The column temperature was maintained at 25 °C and

Detector response (mV) the injection volume was 5 μL. The LC– MS method is not quantitative because 40 Poly TAG DAG FFA of the lack of pure standards. In our 20 22 24 26 28 30 method we used cholesterol-D6 as the Retention time (min) internal standard enabling us to accurately establish (relative) concentration Figure 3: Determination of the relative content of polytri-, tri-, di-, monoacylglycerides, and free values. fatty acids by size-exclusion chromatography. Size-Exclusion Chromatography The HPLC system consisted of a Shi- madzu LC-10 isocratic pump equipped IS with an Optimas autosampler (Sep- arations/Spark Holland), a GT-103 solvent degassing unit (Separations/ Spark Holland), a Mistral column oven (Separations/Spark Holland), and a Shodex RI-71 refractive index detector (Shodex). Four 300 mm × 7.5 mm, 5-μm PLgel polystyrene–divinylben- IS

Abundance (%) zene size-exclusion chromatography (SEC) columns (Agilent) connected in series were used for the separation. The compounds were eluted at a flow rate of 0.8 mL/min using tetrahydrofuran as eluent at isocratic pump conditions. The 25 30 35 40 45 50 55 60 column temperature was maintained Retention time (min) at 30 °C and the injection volume was Figure 4: Normal-phase LC–MS analysis of fractions isolated using size-exclusion chromatography. 10 μL. Chromeleon Chromatography Top: Fraction of TAGs and compounds of similar molecular size. Bottom: Fraction of DAGs and software (Interscience) was used for compounds with similar molecular size. data acquisition.

(purity 97–98%) was purchased from was an Agilent 6410 triple-quadrupole Volatile Compounds by GC Cambridge Isotope Laboratories for use MS system equipped with an Agilent Hexanal measurements were performed as an internal standard. Acetonitrile, G1971A APPI source. The ion source according to AOCS recommended prac- isopropyl alcohol (IPA), and n-hexane was equipped with a krypton discharge tice Cg 4-94 (10) on an Agilent 7890A (ULC–MS-grade) were obtained from lamp and was operated in positive ion- GC–MS system equipped with a Gerstel Biosolve and tetrahydrofuran (HPLC- ization mode. The optimized gas and MPS2XL multipurpose sampler. grade) was purchased from Merck. vaporizer temperatures were 250 °C and 275 °C, respectively. The gas flow Results and Discussion Normal-Phase LC–APPI-MS was 7 L/min, the nebulizer pressure was The stability of various mayonnaise The HPLC system consisted of an Ag- 40 psi, the capillary voltage was 2 kV, formulations was studied in a shelf- ilent 1200SL binary pump equipped and the fragmentor voltage was 80 V. life test at ambient conditions over with an HTC PAL autosampler (CTC Full scan mass spectra were acquired 200 days. The samples studied were Analytics), an Agilent 1200 series micro from m/z 250 to 1000. mayonnaises obtained from local su- vacuum degasser, and an Agilent 1200 A 250 mm × 3.0 mm, 3-μm pursuit permarkets and prototypes contain- series thermostated column compart- XRs 2 Diol column (Varian) was used ing natural antioxidants (confiden- ment. The mass spectrometer used for separation. The analytes were eluted tial stabilization technology 1 and 2).

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Epoxy–Oxo lipids 897.7 603.5

381.3 619.5 493.3 879.6 661.5 441.3 577.4 675.5 927.7 467.3 757.5 955.7

827.5 2½-glycerides

493.3 293.2 717.5 787.5

505.4 277.2 373.2 603.4 662.6 841.5 548.2 617.5 911.6

897.7 Hydroperoxides 881.7 375.4 MS response 493.4 603.5 855.6 619.5 757.5 913.7 507.3 577.4 797.6

603.5 Highly oxidized lipids 493.4

927.7 533.4 827.6 943.6 617.5 771.5 869.6 663.5 397.4 507.4 577.5

300 400 500 600 700 800 900 1000 Mass-to-Charge (m/z)

Figure 5: Mass spectra of lipid oxidation products after removal of abundant diacylglycerols.

For each formulation tested and each directly injected into the normal-phase 40-min to 50-min time interval, tri- time-point, 1 mL of sample was put in LC–APPI-MS instrument. glycerides with a single hydroperoxide a 20-mL headspace vial. The vial was The normal-phase LC separation is group are eluted. More oxidized lipids filled with air, capped, and placed in a largely based on polarity, with retention are retained longer, with lipids contain- temperature-controlled room (20 °C) of the lipids and their oxidation prod- ing two or more hydroperoxide groups in the dark. Samples were taken at sev- ucts being determined by the presence eluting at retention times above 50 min. en-day intervals and were immediately of polar groups resulting from the oxi- APPI is known for its efficient ion- stored at -80 °C to stop any further dation reaction. The retention order of ization of nonpolar or low-polarity oxidation and awaited analysis as one the oxidation products was established compounds, such as sterols and their large batch at the end of the shelf-life in our earlier studies (9). As an illustra- oxidation products (11). In APPI the study. Preparation of the samples con- tion, Figure 1 shows the normal-phase ion source is less susceptible for pol- sisted of freeze-drying for 48 h. Water LC–APPI total ion current chromato- lution because no additives are needed was removed under mild conditions gram of a 154-day aged mayonnaise to facilitate ionization as would be the and all species, irrespective of whether after freeze-drying. The first eluting case in electrospray ionization. In pho- they were present in the water- or lip- group are the unreacted TAG, which toionization, two ionization pathways id-phase, or at the oil-droplet interface, are eluted almost unretained. The can be exploited: direct photoioniza- were retained. After freeze-drying, next group is that of the epoxy- or oxo- tion or dopant-assisted photoioniza- 4 mL of n-hexane was added to the TAG, oxidation products containing tion (12). In this work ionization of vials to dissolve the lipids and their ox- one additional oxygen as a ketone or the compounds was achieved by APPI idation products. Upon dissolution the aldehyde functionality. 2½ glycerides using the self-doping effect of the hex- proteins precipitated and clear sample are slightly more polar and are eluted ane-based mobile phase (13). The pho- solutions were obtained that could be between 25 min and 40 min. In the tons emitted by the krypton discharge

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Ambient shelf-life

Hydroperoxides Oxo-2½ glycerides

0 50 100 150 200 0 50 100 150 200 Highly oxidized lipids Epoxides Relative abundance

0 50 100 150 200 0 50 100 150 200 Storage time (days)

EDTA No stabilizer Technology 1 Technology 2

Figure 6: Trend lines of nonvolatile lipid oxidation markers measured during an ambient shelf-life test. Different lines represent different stabilization technologies.

lamp have energies in the range of 10 seem to be present. Mass losses of 18 greatly aid the interpretation of the re- to 10.6 eV, which is above the ioniza- and 34 Da are seen, indicating the loss sults obtained. tion potential of the n-hexane eluent of water or hydroperoxide groups and In the initial normal-phase LC sep- (10.18 eV). Via charge exchange and resulting in the fragment ions [M+H- arations of the aged mayonnaise, mul- + + + proton transfer, the primary hexane H2O] or [M H-H2O2] , respectively. tiple peaks were seen with polarities ions cause ionization of the analytes in Smaller ions with m/z values in the corresponding to one or more hydrop- the gas phase. The effectiveness of this 400 Da to 600 Da range are also seen eroxy-groups, yet molecular weights approach is shown in Figure 2. This fig- in this retention time window. These far below that of the HOO-TAG. SEC ure shows the mass spectra for some of could result from in-source fragmen- was applied to determine the molecular the more abundant peaks in the time tation, but they could also represent weight distribution of the sample, fo- window from 40 to 50 min in Figure other species of similar polarity and cusing in particular on the polymerized 1. All mass spectra show intense sig- lower molecular weight. Experiments TAG, the TAG, as well as the di- and nals, especially in the mass range be- at other ionization settings (fragmentor mono-acylglycerols and the free fatty tween m/z 850 and 900. From the LC voltage 50 V to 100 V) did not change acids. By using SEC with refractive elution positions it is likely that these the abundance of the 400–600 Da ions. index detection the relative content of signals represent hydroperoxides. The It was concluded that these signals are each of these individual groups could molecular ions of hydroperoxide-TAGs not caused by fragmentation, but most be determined. A representative SEC with three C18 fatty acyl chains would likely represent other compounds. chromatogram is shown in Figure 3. be expected at m/z values of approxi- Clearly the chromatograms and MS The largest peak represents the TAGs, mately 920 Da, depending on the num- spectra obtained are too complex for or, more correctly phrased, TAGs and ber of double bonds in the molecule. further interpretation. Additional sep- compounds with a molecular size sim- However, these molecular ions do not aration of these complex samples could ilar to that of a TAG. This group ac-

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Accelerated shelf-life

Hydroperoxides Oxo-2½ glycerides

0 10 20 30 40 0 10 20 30 40

Poly-triglycerides Hexanal 10.0 Relative abundance 7.5

% 5.0

2.5

0.0 0 10 20 30 40 0 10 20 30 40 Storage time (days) EDTA No stabilizer Technology 1 Technology 2

Figure 7: Trend lines of volatile and nonvolatile oxidation markers measured during a 50 °C accelerated shelf-life test. Different lines represent different stabilization technologies.

counts for 95.8% of the total signal. Pri- the analysis of samples from the initial The total ion current chromato- mary oxidation products are just one or stages of oxidation where the levels of grams of the size fractions containing a few oxygen atoms larger than the TAG the oxidation products are still low. the (oxidized-)TAGs and the DAGs are and hence in SEC are coeluted with the To eliminate overlap of DAGs and shown in Figure 4. Most of the polar TAGs. Significant levels of diglycerides FFAs with the oxidized TAG in the nor- compounds in both fractions are eluted (DAGs) and free fatty acids (FFAs) were mal-phase LC–APPI analysis, samples between 40 min and 50 min. Note that also seen in the SEC separation. These were prefractionated based on size by the nonoxidized TAGs are eluted al- species are present at concentration lev- SEC prior to normal-phase LC analysis. most unretained in normal-phase LC els of 2.4% and 0.7%, respectively, and Fractions of the eluent were collected in and are not shown in these chromato- could explain the low-molecular-weight amber colored vials in four time win- grams. Hence, the peaks seen in the polar species seen in normal-phase LC– dows: 29–33.5 min, poly-TAG window, size fraction of the TAG are oxidized APPI-MS. Furthermore, polymerized 33.5–35.5 min, (oxidized-)TAG, 35.7– compounds with a size comparable to triglycerides are present at a concentra- 37.5 min, DAG, and 37.8–40 min, FFA. that of the TAG. The normal-phase tion level of 1.1%. These are secondary To obtain enough material, the proce- LC–MS profiles of the two SEC frac- oxidation products known to be formed dure was repeated twice and the fractions containing the (oxidized-)TAG from the fusion of two hydroperoxides tions were combined. After evaporation or the smaller DAG are very different. via different pathways (14). The levels of the eluent under a stream of nitrogen, Clearly, however, without SEC presep- of DAGs and FFAs are high relative to the fractions were redissolved in 100 μL aration, these two compound groups of those of the oxidized TAGs. They in- n-hexane containing cholesterol D6 as widely different size yet identical polar- terfere with the TAG-oxide identifica- internal standard before normal-phase ity would overlap. Six main peaks can tion and quantification, especially in LC–APPI-MS analysis. be distinguished in the DAG fraction.

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Table I: Lipid oxidation products in mayonnaise with corresponding retention time and m/z windows. The molecular structures indicated are typical examples. R represents the fatty acid chains. Chain length can vary depending on the origin of the oil. Start RT End RT Minimum m/z Maximum m/z Epoxy–Oxo lipids 10 22 891 900

O O CH O O 3

O R1

O R2 O

Oxo-2½ glycerides 23 35 821 830 O O

O O

O R1

O R2 O Hydroperoxides 40 50 891 900

O OOH

O O CH3

O R1

O R2 O Highly oxidized lipids 50 58 938 950

O OOH CH O O 3

O CH3 OOH O O

The first eluted peak at 42 min has an are not shown in Figure 4. More-polar no unsaturation (OS-epoxide) (14). All m/z of 603.5, the second peak an m/z of species, such as the epoxy-TAGs and the TAGs with two adjacent O fatty acids 601.5 (43 min), and the third peak has hydroperoxy-TAGs, are eluted in the re- can form this fragment ion. In the an m/z 599.5 (44 min). These masses tention time windows of 10–25 min and rape seed oil used to prepare the may- + + correspond to the [M H-H2O] ions 40–50 min. The interferences of the un- onnaises analyzed here the primary of the diacylglycerols OO, OL, and LL/ reacted DAG are now removed meaning TAGs showing this behavior are OOLn, OLn, respectively, where O represents that the mass spectra are easier to inter- OOO, and POO. The fragment ion at oleic acid, L linoleic acid, and Ln lino- pret. In Figure 5, MS spectra are shown m/z 493.4 is formed from breakage of lenic acid. Positional isomers of these of the epoxy–oxo lipids, hydroperoxides, this epoxide bond, resulting in the loss

diacylglycerols also are eluted in the 2½ glycerides, and the highly oxidized of a C9H18 group. same retention order between 47 min lipids. The MS spectra of some import- The earlier eluted epoxides yield and 49 min. From this it can be con- ant classes of lipid oxidation products mainly protonated molecular ions, with cluded that the low-molecular-weight are discussed in detail below. an example of this being m/z 897.7. The peaks seen in the initial normal-phase For the hydroperoxides, protonated mass spectrum is nearly identical to LC separation without prior SEC sepa- molecules [M+H]+ are visible, for ex- that of the hydroperoxides, except that ration were not representing oxidized ample at m/z 913.7, at a relatively low there is no mass loss of 34 Da visible

TAGs, but rather partial hydrolysis abundance. The most prominent peaks (loss of H2O2). products formed upon oxidation. The are m/z 897.7 and 883.7, and are formed The 2½ glycerides oxidation prod- retention time differences between the by the loss of water giving [M+H- ucts, the remaining nonvolatile part + different DAGs can be explained by the H2O] , or the loss of a hydroperoxide of the triglyceride after chain-scission + + number of double bonds and the po- group, leading to [M H-H2O2] ions. and release of the volatile off-smell, are sition of the free hydroxide group on The DAG fragment ion m/z 603.5 is secondary oxidation products formed the glycerol backbone. The last eluted formed after loss of the fatty acyl chain from breakdown of the hydroperoxides. peaks have a vacant outer position on with the hydroperoxide functionality. They are present in the DAG fraction, the glycerol backbone. Another characteristic DAG fragment but not clearly visible in the TIC chro- The “triglyceride” SEC fraction con- ion is visible at m/z 619.6. During ion- matogram. Their abundance is low in tains the triglycerides next to the oxi- ization water is lost and an epoxide comparison to the signal of the DAGs. dized TAGs. The unreacted TAGs are group is formed across the site of un- The structures with 2½ fatty acid unretained in normal-phase LC and saturation converting it to a chain with chains are actually oxo-2½ glycerides,

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the levels and types of oxidation products. Figure 6 shows the response plots of the nonvolatile species determined by normal-phase LC–APPI-MS. Over Triglycerides Epoxy–Oxo Oxygeny 150 samples could be analyzed without maintenance. From this perspective the newly developed APPI-MS method performed much better than our previously described ESI approach (9). Par- allel to the LC–MS measurements sen- Highly oxidized Hydroperoxides sory testing was performed. Hexanal measurements were also performed, but the levels were too low for the classical static headspace method. Clear trends are visible in the plotted data, and the Oxo-2½ levels of all monitored oxidation markers increase with storage time. More- Polymerized lipids Volatiles over, differences between the applied stabilization technologies are visible Figure 8: Schematic representation of the “oxidation engine,” a continuous process where as well. The formulation with EDTA volatile, nonvolatile, and oxidation end products are formed through routes with different (positive control) appears to function intermediates. best in delaying oxidation by showing the lowest increase in NONVOLLOPS with molecular masses corresponding with similar polarity but different sizes. over time. When no stabilization tech- to that of the parent triglyceride (for The combined results of the chromato- nique was applied (negative control), example, LLO) after the loss of a hex- graphic and MS spectral evaluation of the oxidation products increased the

anal molecule (C6H12O mass 100 Da). the different lipid oxidation groups are most rapidly. The proprietary stabi- It is important to note that the oxo-2½ presented in Table I. The retention time lization technology 2 performs better glycerides group is very complex. The windows and the m/z values used to in this shelf-life test than technology 1: original molecules can contain differ- generate extracted ion chromatograms formation of the secondary oxidation ent fatty acids from which different to zoom in on specific oxidation prod- products oxo-2½ glycerides and highly volatiles can be split off. Moreover, ucts can be seen. Examples of the mo- oxidized lipids occurs at a later time- the remaining fatty acid chains can lecular structures of the oxidized spe- point in the shelf-life test and the levels be oxidized at one or more additional cies are also given in the table. Specific are lower. Interestingly, hydroperox- positions. In the extracted ion chro- positions of the polar oxidation groups ides seem to reach a constant plateau matograms, the oxo-2½ glycerides are on the fatty acid chains can vary de- level, more or less identical for each of detectable in the samples. The oxo-2½ pending on the original location of the the formulations tested. The possibility glycerides with an additional oxidation double bonds. Note that the combina- that this phenomena was caused by a group are eluted later from the column tion of retention times and spectral in- nonlinear response of the MS detector and do not interfere with the analysis. formation is unique. This means that was ruled out by measurement of sev- As the degree of oxidation increases not all samples have to go through the eral diluted samples yielding similar and TAG with multiple oxygens incor- time-consuming SEC prefractionation. curves. The occurrence of a plateau porated in their structure are formed, Analyzing a few samples after DAG re- suggests that the formation of hydrop- the mass spectra become even more moval is sufficient to establish the time eroxides at some point comes to a halt, complex. Possible structures are ep- windows and MS traces of interest. or that the rate of breakdown and for- oxyhydroperoxides, dihydroperoxides, To meet the clear trend in society for mation become equal. The latter expla- dihydroxyperoxides, and cyclic perox- more natural foods, the food industry is nation here is much more logical. The ides. In the mass spectra multiple losses trying to replace non-natural stabiliza- hydroperoxides are unstable interme- of 18 and 34 Da are visible, suggesting tion strategies by natural preservation diates that can be converted into other the loss of water and hydroperoxide routes. To this end formulations with volatile and nonvolatile products. This groups upon ionization. Full interpre- differences in the applied stabilization is supported by the steady increase of tation of these compounds remains dif- technology were tested in a large-scale oxo-2½ glycerides, degradation prod- ficult, however. storage test. The main goal of this study ucts of the hydroperoxides, over time. The SEC fractionation was applied was to find suitable replacements for Attempts to correlate the occurrence of on a limited number of samples to EDTA (E385, E386) and understand nonvolatile oxidation products in this build understanding of the MS spectra how the different stabilization routes ambient shelf-life trial with the release by eliminating overlapping compounds determine the oxidation reactions and of hexanal failed because of an insuffi-

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cient sensitivity of the classical GC–MS undergo a continuous process of oxi- (4) M. Hicks and J.M. Gebicki, Anal. Bio- headspace method. dation and breakdown and eventually chem. 99(2), 249–253 (1979). To enable correlation of NONVOL- end up as volatiles and polymerized lip- (5) A. Lips, R.A. Chapman, and W.D. Mc- LOPS levels with hexanal release, an ids in the samples. Clearly the reactions Farlane, Oil Soap 20(11), 240–243 additional (accelerated) shelf-life test occurring in lipid oxidation are more (1943). was performed at 50 °C with a sampling complex than the “oxidation engine” (6) H. Ohkawa, N. Ohishi, and K. Yagi, interval of three days. The effect of the suggests (3), yet the principle of the Anal. Biochem. 95(2), 351–358 increased temperature on the formation engine clearly illustrates the processes (1979). of volatile and nonvolatile oxidation occurring. Stabilization technologies (7) B. Barriuso, I. Astiasarán, and D. products is shown in Figure 7. GC–MS control the initial rate of formation of Ansorena, Eur. Food Res. Technol. was applied to measure hexanal (10). hydroperoxides, as well as the speed of 236(1), 1–15 (2013). The SEC method originally set up to the engine in a way still largely not un- (8) S. Panseri, S. Soncin, L.M. Chiesa, remove interfering species was applied derstood. The new methods presented and P.A. Biondi, Food Chem. 127(2), to monitor the formation of polymer- here shed some light on the oxidation 886–889 (2011). ized TAG. reactions and products forming, and (9) L. Steenhorst-Slikkerveer, A. Louter, At the 50 °C elevated shelf life tem- can help in making the search for al- H.-G. Janssen, and C. Bauer-Plank, J. perature substantial oxidation oc- ternative stabilization technologies to Am. Oil Chem. Soc. 77(8), 837–845 curred. After 40 days of storage, the protect lipid-rich foods from oxidation (2000). SEC measurements show poly-tri- less of a trial-and-error exercise. (10) AOCS Recommended Practice Cg 4-94. glycerides at up to 10% of the total lipid Volatiles (VOC) in Fats and Oils by GLC. content. In the GC–MS experiments Conclusions Official Methods and Recommended hexanal was clearly detectable. When The newly developed normal-phase LC– Practices of the AOCS, (American Oil looking at the time curves of the hy- APPI-MS method allows the detection Chemists Society, Champaign, Illinois, droperoxides and oxo-2½ glycerides a of nonvolatile lipid oxidation products 1998). trend similar to that seen in the am- in the initial and later stages of lipid ox- (11) C.H. Grün and S. Besseau, J. Chro- bient study is observed, albeit that the idation. With the detailed information matogr. A 1439, 74–81 (2016). oxo-2½ glycerides are detectable much on the NONVOLLOPS, we were able (12) D.B. Robb, T.R. Covey, and A.P. Bru- earlier in the 50 °C storage test. Already to determine the efficiency of different ins, Anal. Chem. 72(15), 3653–3659 after 11 days this marker is detectable stabilization technologies in complex (2000). and clear differences between the for- food emulsions. This was achieved by (13) T. Ghislain, P. Faure, and R. Michels, mulations become apparent. This dif- successful ionization of NONVOLLOPS J. Am. Soc. Mass Spectrom. 23(3), ferentiation is not so clear from the using the self-dopant effect of the mo- 530–536 (2012). results of the hexanal and poly-TAG bile phase. Detailed interpretation of (14) E.N. Frankel, J. Am. Oil Chem. Soc. analyses in the initial stages of the MS spectra was possible using pre-sep- 61(12), 1908–1917 (1984). oxidation process. Both hexanal and aration by SEC. The relative levels of poly-TAGs show a clear lag phase. Ini- important NONVOLLOPS such as hy- Boudewijn Hollebrands, tially they are hardly formed, but after droperoxides, oxo-2½ glycerides, and MSc, completed his master study in a certain lag phase their levels start epoxides could be determined in large analytical sciences at the University of to increase at a much faster rate than sample sets from ambient and acceler- Amsterdam in 2013. Currently, he is a those of the oxo-2½ glycerides. From ated stability tests. It is envisaged that research scientist in the analytical depart- this observation, we postulate that the the developed method will play an im- ment of Unilever R&D Vlaardingen, the oxo-2½ glycerides may undergo further portant role in developing new, natural Netherlands. His work focuses on the breakdown and are, like the hydroper- stabilization technologies to suppress development of hyphenated chromato- oxides, not end products of the oxida- lipid oxidation in lipid-rich foods. graphic techniques and high-resolution tion reaction. Whether they are lost mass spectrometry methods for food and by conversion to poly-TAG or oxidize Acknowledgments nutritional analysis. further by the additional formation The authors thank Justin van ‘t Veer of hydroperoxide or epoxide groups for performing the GC–MS hexanal Hans-Gerd Janssen is science lead- on the remaining unreacted fatty acid analysis. er of chromatography and mass spectrom- chains cannot be derived from the cur- etry at Unilever R&D Vlaardingen. He also is rent data. This concept of continuous References a part-time professor in biomacromolecular oxidation with intermediates and end (1) G. Billek, Eur. J. Lipid Sci. Technol. analysis at the University of Amsterdam, products is schematically illustrated in 102(8–9), 587–593 (2000). Amsterdam, the Netherlands. Professor Figure 8. In this “oxidation engine,” tri- (2) J. Kanner, Mol. Nutr. Food Res. 51(9), Janssen’s research interests include the glycerides and oxygen are the starting 1094–1101 (2007). development of multidimensional separa- products that form intermediate oxi- (3) H. Yin, L. Xu, and N.A. Porter, Chem. tion systems for food, biomedical, and envi- dation products. These products may Rev. 111(10), 5944–5972 (2011). ronmental analysis. ◾

black ES1048057_LCGCSUPP0518_033.pgs 05.01.2018 15:24 ADV 34 Current Trends in Mass Spectrometry May 2018 chromatographyonline.com PRODUCTS & RESOURCES Solvent evaporators HPLC system Biotage’s TurboVap LV II and EH C-Vue Chromatography’s compact solvent evaporators are CH2 HPLC system is designed with enhanced visibility, designed to provide removable and adjustable nozzles, separations with UV-ab- exchangeable manifolds, evapora- sorbance detection or to tion flow gradients, and a touch- be used as a front end screen surface. The EH system for mass spectrometers. reportedly is integrated with the According to the company, company’s Extrahera automated the system can be used sample preparation system, and is as a benchtop laboratory instrument or as a portable instrument for aesthetically similar. field use. Biotage, LLC, C-Vue Chromatography, Charlotte, NC. Simpsonville, SC. www.biotage.com www.c-vuelc.com

Pyrolyzer Mass spectrometer The Pyprobe 6000 pyro- PerkinElmer’s QSight Triple Quad LC–MS/ lyzer from CDS Analytical MS mass spectrometer is designed for is designed with a pyrol- applications such as food safety, environ- ysis sample tube that is mental testing, and industrial research. gravity-dispensed into the According to the company, the mass system’s Drop-In-Sample- spectrometer provides high sensitivity Chamber (DISC). According and uptime, and offers remote support to the company, the RSD capabilities. results are within 2% due to PerkinElmer, the positioning of the sample Waltham, MA. in the pyrolysis coil. www.perkinelmer.com/qsight CDS Analytical LLC, Oxford, PA. www.cdsanalytical.com

E lectron multipliers Hydrogen gas generator Discrete dynode electron Proton OnSite’s G600-HP high- multipliers from Photonis are purity hydrogen gas designed as plug-and-play generator is designed with replacements for ICP-MS systems. proton exchange membrane The electron multipliers reportedly technology to produce hydrogen provide a mass range to 238U for a variety of laboratory and gating of less than 6 ns. applications. According to According to the company, nearly the company, the generator 200 electron multiplier types are produces hydrogen carrier gas at available for current and legacy 99.99999% purity. instruments. Proton OnSite, Photonis USA, Wallingford, CT. Sturbridge, MA. ww2.protononsite.com/LCGC/ www.photonis.com CTMS/PR

HILIC columns SEC–MS application note HILIC pac VC-50 columns An application note from Tosoh Bio- from Shodex are designed science titled “Size-Exclusion Chroma- for hydrophilic interaction tography/Mass Spectrometry (SEC/ chromatography analysis MS) Analysis of a Bispecific Antibody” of monoamine type neu- reportedly describes a bispecific T cell rotransmitters and oral engager (BiTE) consisting of two linked anti-diabetic drugs. Accord- single-chain variable fragments that ing to the company, the was analyzed by SEC–MS using a TSK- columns are packed in PEEK gel UP-SW3000, 2-μm column. to decrease interactions Tosoh Bioscience LLC, between compounds and King of Prussia, PA. housing sides and are suitable for LC–MS analysis. www.tosohbioscience.com Shodex/Showa Denko America, Inc., New York, NY. www.shodexhplc.com

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Head Analysis of Polyethylene by Pyrolysis-GC×GC–MS

Daniela Peroni, JSB

Pyrolysis-gas chromatography–mass spectrometry (py-GC–MS) is a very useful technique for polymer characterization. The careful- ly-controlled pyrolysis heating step provides informative and specif c products that can be separated and identif ed by GC–MS to estimate the polymeric composition/structure. However, these pro- f les are sometimes too complex to be characterized properly by conventional GC, leading to loss of information. Figure 2: 3D view of the py-GC×GC–MS chromatogram of PE. Comprehensive two-dimensional gas chromatography (GC×GC) provides enhanced resolving power and peak capacity by com- bining two different separation mechanisms in one analysis. This allows for more detailed separations and more complete characterization of complex matrices. Here we show the py-GC−MS and py- GC×GC–MS prof les of polyethylene (PE) to prove the advantages arising from coupling pyrolysis and GC×GC. Figure 1 shows the GC–MS chromatogram of a PE sample subject to pyrolysis at 750 °C and separated on a non-polar column. The characteristic PE pyrolysis prof le shows a repeating unit of triplets of paraff ns (diene-alkene-alkane) for every carbon number. Be- tween these major peaks, there are a number of smaller, unresolved peaks commonly identif ed as branched paraff ns. The two-dimensional (2D) separation of the same pyrolysis profile Figure 3: Py-GC×GC–MS chromatogram of PE. is shown in Figure 2. The second dimension separation based on polarity grants additional composition information. Several peaks are the case of naphthalene and C12 triplet. In the 2D plot they are fully visible between the triplets, providing a better idea of the number of separated and show clean MS spectra that allow for easy identification. branched paraffins present. Additionally, there are a number of polar compounds more retained on the second dimension (Figure 3). These Instrumentation analytes are not visible at all in the GC–MS analysis due to their small CDS Pyroprobe coupled to an Agilent 7890B GC equipped with a abundance and the co-elution with the aliphatic components, like in Zoex ZX2 cryogen-free thermal modulator and a 5977A Agilent Mass Selective Detector (MSD).

In conclusion, Py-GC–MS analysis of polyethylene shows a characteristic pattern of paraff n triplets for every carbon number. However, separation is not suff cient to unravel the sample complexity. Characterization obtained by coupling pyrolysis with GC×GC provides more information. Several aromatic compounds are identif ed in the 2D pyrogram of polyethylene which are not detected in the mono-dimensional separation.

CDS Analytical 465 Limestone Rd., P.O. Box 277, Oxford, PA 19363-0277 Figure 1: Py-GC–MS chromatogram of PE. The zoom-ins show a de- tel. (610) 932-3636, fax (610) 932-4158 tail of the prof le and an example of a complex area. Website: www.cdsanalytical.com

magentablackcyanyellow ES1048055_LCGCSUPP0518_035.pgs 05.01.2018 15:23 ADV 36 Medical/Biological ADVERTISEMENT

Analysis of Fentanyl and Its Analogues in Human Urine by LC–MS/MS Shun-Hsin Liang and Frances Carroll, Restek Corporation

Abuse of synthetic opioid prescription painkillers such as fentanyl, along with a rapidly growing list of illicit analogues, is a signif cant public health problem. In this study, we developed a simple dilute-and-shoot method that provides a fast 3.5-min analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC–MS/MS using a Raptor Biphenyl column.

n recent years, the illicit use of synthetic opioids has skyrocketed, Iand communities worldwide are now dealing with an ongoing epidemic. Of the thousands of synthetic opioid overdose deaths per year, most are related to fentanyl and its analogues. With their very high analgesic properties, synthetic opioid drugs such as fentanyl, alfentanil, remifentanil, and sufentanil are potent painkillers that have valid medical applications; however, they are also extremely 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 addictive and are targets for abuse. In addition to abuse of these Time (min) prescription drugs, the current opioid crisis is fueled by a growing Figure 1: The Raptor Biphenyl column effectively separated all number of illicit analogues, such as acetyl fentanyl and butyryl fen- target compounds in urine with no observed matrix interferences.

tanyl, which have been designed specif cally to evade prosecution Peak elution order: norfentanyl-D5, norfentanyl, remifentanil, acetyl 13 by drug enforcement agencies. fentanyl- C6, acetyl fentanyl, alfentanil, fentanyl-D5, fentanyl, carfentanil-D , carfentanil, butyryl fentanyl, sufentanil-D , sufentanil. As the number of opioid drugs and deaths increases, so does 5 5 the need for a fast, accurate method for the simultaneous analysis method provides accurate, precise identif cation and quantitation of of fentanyl and its analogues. T erefore, we developed this fentanyl and related compounds, making it suitable for a variety of LC–MS/MS method for measuring fentanyl, six analogues, and one testing applications, including clinical toxicology, forensic analysis, metabolite (norfentanyl) in human urine. A simple dilute-and-shoot workplace drug testing, and pharmaceutical research. sample preparation procedure was coupled with a fast (3.5 min) chromatographic analysis using a Raptor Biphenyl column. T is Experimental Conditions Table I: Analyte transitions Sample Preparation Precursor Product Ion Product Ion Analyte Internal Standard Ion Quantif er Qualif er T e analytes were fortif ed into pooled human urine. Norfentanyl 233.27 84.15 56.06 Norfentanyl-D An 80 μL urine aliquot was mixed with 320 μL of 5 70:30 water–methanol solution (f vefold dilution) Acetyl fentanyl 323.37 188.25 105.15 Acetyl fentanyl-13C 6 and 10 μL of internal standard (40 ng/mL in meth- Fentanyl 337.37 188.26 105.08 Fentanyl-D 5 anol) in a T omson SINGLE StEP f lter vial (Restek Butyryl fentanyl 351.43 188.20 105.15 Carfentanil-D5 cat. #25895). After f ltering through the 0.2 μm PVDF

Remifentanil 377.37 113.15 317.30 Norfentanyl-D5 membrane, 5 μL was injected into the LC–MS/MS.

Sufentanil 387.40 238.19 111.06 Sufentanil-D5 Carfentanil 395.40 113.14 335.35 Carfentanil-D Calibration Standards 5 and Quality Control Samples Alfentanil 417.47 268.31 197.23 Acetyl fentanyl-13C 6 T e calibration standards were prepared in pooled hu- Norfentanyl-D 238.30 84.15 Ñ Ñ 5 man urine at 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, Acetyl fentanyl-13C 329.37 188.25 Ñ Ñ 6 10.0, 25.0, and 50.0 ng/mL. T ree levels of QC sam-

Fentanyl-D5 342.47 188.27 Ñ Ñ ples (0.75, 4.0, and 20 ng/mL) were prepared in urine for testing accuracy and precision with established cal- Sufentanil-D5 392.40 238.25 Ñ Ñ ibration standard curves. Recovery analyses were per- Carfentanil-D5 400.40 340.41 Ñ Ñ

magentablackcyanyellow ES1048071_LCGCSUPP0518_036.pgs 05.01.2018 15:30 ADV ADVERTISEMENT Medical/Biological 37

formed on three dif erent days. All standards and QC samples monitored for both research and abuse. Using 1/x weighted linear were subjected to the sample preparation procedure described. regression (1/x2 for butyryl fentanyl), calibration linearity ranged LC–MS/MS analysis of fentanyl and its analogues was from 0.05 to 50 ng/mL for fentanyl, alfentanil, acetyl fentanyl, performed on an ACQUITY UPLC instrument coupled with butyryl fentanyl, and sufentanil; from 0.10 to 50 ng/mL for a Waters Xevo TQ-S mass spectrometer. Instrument conditions remifentanil; and from 0.25 to 50 ng/mL for norfentanyl and were as follows, and analyte transitions are provided in Table I. carfentanil. All analytes showed acceptable linearity with r2 values of 0.996 or greater and deviations of <12% (<20% for the lowest Analytical column: Raptor Biphenyl (5 μm, concentrated standard). 50 mm × 2.1 mm; cat. #9309552) Guard column: Raptor Biphenyl EXP guard column Accuracy and Precision cartridge, (5 μm, 5 mm × 2.1 mm; Based on three independent experiments conducted on multiple cat. #930950252) days, method accuracy for the analysis of fentanyl and its ana- Mobile phase A: 0.1% Formic acid in water logues was demonstrated by the %recovery values, which were Mobile phase B: 0.1% Formic acid in methanol within 10% of the nominal concentration for all compounds at Gradient Time (min) %B all QC levels. T e %RSD range was 0.5–8.3% and 3.4–8.4% 0.00 30 for intraday and interday comparisons, respectively, indicating 2.50 70 acceptable method precision (Table II). 2.51 30 3.50 30 Conclusions Flow rate: 0.4 mL/min A simple dilute-and-shoot method was developed for the quan- Injection titative analysis of fentanyl and its analogues in human urine. volume: 5 μL T e analytical method was demonstrated to be fast, rugged, and Column temp.: 40 °C sensitive with acceptable accuracy and precision for urine sample Ion mode: Positive ESI analysis. T e Raptor Biphenyl column is well suited for the analysis of these synthetic opioid compounds and this method can be Results applied to clinical toxicology, forensic analysis, workplace drug Chromatographic Performance testing, and pharmaceutical research. All eight analytes were well separated within a 2.5-min gradient elution (3.5-min total analysis time) on a Raptor Biphenyl column (Fig- ure 1). No signif cant matrix interference was observed to negatively af ect quantif cation of the f vefold diluted urine samples. T e 5-μm particle Raptor Biphenyl column used here is a superf cially porous particle (SPP) column. It was selected for this method in part because it provides similar performance to a smaller particle size fully porous particle (FPP) column, but it generates less system back pressure. Restek Corporation Linearity 110 Benner Circle, Bellefonte, PA 16823 Linear responses were obtained for all compounds and the tel. 1 (814) 353-1300 calibration ranges encompassed typical concentration levels Website: www.restek.com

Table II: Accuracy and precision results for fentanyl and related compounds in urine QC samples

QC Level 1 (0.750 ng/mL) QC Level 2 (4.00 ng/mL) QC Level 3 (20.0 ng/mL)

Average Conc. Average % Average Conc. Average % Average Conc. Average % Analyte %RSD %RSD %RSD (ng/mL) Accuracy (ng/mL) Accuracy (ng/mL) Accuracy Acetyl fentanyl 0.761 102 1.54 3.99 99.7 2.08 19.9 99.3 0.856 Alfentanil 0.733 97.6 3.34 3.96 98.9 8.38 20.9 104 6.73 Butyryl fentanyl 0.741 98.9 6.29 3.77 94.3 6.01 20.8 104 4.95 Carfentanil 0.757 101 7.34 3.76 94.0 4.64 20.6 103 4.24 Fentanyl 0.761 102 1.98 3.96 99.1 2.31 19.9 99.6 1.04 Norfentanyl 0.768 103 6.50 4.04 101 1.84 20.1 101 2.55 Remifentanil 0.765 102 3.42 3.97 99.2 3.68 20.8 104 4.14 Sufentanil 0.752 100 1.67 3.93 98.3 1.28 20.1 100 0.943

magentablackcyanyellow ES1048068_LCGCSUPP0518_037.pgs 05.01.2018 15:31 ADV 38 Current Trends in Mass Spectrometry May 2018 chromatographyonline.com

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