Artefact Peaks Research Articles

Generic multicriteria approach to determine the best precipitation agent for removal of biomacromolecules prior to non-targeted metabolic analysis

The removal of biomacromolecules from biofluids decreases the sample complexity and lower electrospray suppression effects. Furthermore, it can increase the analysis sensitivity, precision, and selectivity. Often removal approaches evaluate the model based on a single criterion, like protein removed or response of one of few specific metabolites. In this study, we used a multicriteria approach to test the effect of using the solvents methanol and acetonitrile (organic solvent precipitation), trichloroacetic acid (acidic precipitation) and ammonium sulphate (salting out) to remove biomacromolecules from a downstream recovery process from a bacillus fermentation. The downstream recovery process intermediates were analysed using reversed-phase ultra-high-pressure liquid chromatography with electrospray ionisation and high-resolution time-of-flight mass spectrometry detection. To evaluate the pre-treatment agents the following multicriteria was applied i) practical considerations, ii) total amino acid in the precipitated pellet, iii) putative identification of the molecules removed or created by the different treatments, iv) coherence between high quality extracted ion chromatograms (repeatability of DW-CODA) and v) replicate consistency from principal component analysis score values obtained by using the CHEMometric analysis of sections of Selected Ion Chromatograms (CHEMSIC) method. This study presents a generic workflow to find the best pre-treatment for removing bio-macromolecules from biofluids with a multicriteria approach. In our case, the best protein removal strategy for downstream recovery intermediates was acetonitrile precipitation. This method showed high precision, created few artefact peaks compared to simple sample dilution, and mainly removed small peptides.

Challenges with co-amplification of microbial DNA in interpretation of STR profiles obtained from human skeletal remains

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.

Artefact peaks of pore size distributions caused by unclosed sorption isotherm and tensile strength effect

The pore size distribution (PSD) is an important property of the porous materials, but an accurate and reliable PSD is very difficult to obtain. Quenched Solid Density Functional Theory (QSDFT) is widely used to analyze the pore size distribution. Here, a series of unclosed and closed isotherms of activated carbon were measured and compared. The experimental results revealed the appearance of an artefact peak in the PSD for the unclosed isotherms by employing QSDFT equilibrium model. Moreover, the location of the artificial peak is highly related to the end point of the desorption branch. In addition, tensile strength effect (TSE) illustrates the limit of mechanical stability of liquids in pores, and subsequent cavitation generates an artefact peak at around 2.8 nm. Interestingly, it was found that isotherm data associated with TSE can be used to calculate the total pore volume of all large cavities in which cavitation occurs.

Characterizing various monoclonal antibodies with milder reversed phase chromatography conditions

Reversed phase liquid chromatography (RPLC) of therapeutic monoclonal antibodies (mAbs) is often performed at elevated temperatures (80–90 °C) and in the presence of relatively high concentrations of TFA (0.1%). Under such conditions, it is possible to achieve suitable performance in terms of peak shapes and recoveries. Yet, it is also possible, to cause on-column hydrolysis and the generation of artefact peaks. Interestingly, a wide-pore silica-based superficially porous (SPP) material with a high coverage phenyl bonding was recently introduced, and may offer a chance to perform protein RPLC with milder conditions (lower temperatures and lower TFA concentrations). To evaluate this possibility, 23 mAbs approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) were analysed on this new column, as well as a reference C4 SPP widepore silica-based column. Separations were performed at various temperatures ranging from 60 to 90 °C using various proportions of TFA and FA ranging from 0.1% TFA to 0.01% TFA/0.09% FA. It appears that temperature can be reduced down to 75 °C for intact mAbs and 65 °C for mAb subunits, using the high coverage phenyl bonded stationary phase. At such temperatures, desirable peak shape and >90% recovery can be observed for each of the studied mAbs. To achieve the same performance with the reference C4 bonded stationary phase, it was necessary to work at 90 °C and 85 °C, respectively. In addition, for mAb subunit analysis, it has been found that combining the phenyl bonded stationary phase with a 0.03% TFA/0.07% FA mobile phase can yield separations similar those obtained with 0.1% TFA, but with an increase of mass spectrometric sensitivity by about 40%.This work provides examples of milder conditions (65 °C and 0.03% TFA + 0.07% FA) being successfully employed in the RPLC(-MS) analysis of mAb subunits. A suitable stationary phase was selected, and milder conditions allow an improved MS sensitivity, while maintaining comparable elution profiles. Moreover, the use of milder conditions may reduce the risk of artefact peaks.

Retaining both discrete and smooth features in 1D and 2D NMR relaxation and diffusion experiments

A new method of regularization of 1D and 2D NMR relaxation and diffusion experiments is proposed and a robust algorithm for its implementation is introduced. The new form of regularization, termed the Modified Total Generalized Variation (MTGV) regularization, offers a compromise between distinguishing discrete and smooth features in the reconstructed distributions. The method is compared to the conventional method of Tikhonov regularization and the recently proposed method of L1 regularization, when applied to simulated data of 1D spin-lattice relaxation, T1, 1D spin-spin relaxation, T2, and 2D T1-T2 NMR experiments. A range of simulated distributions composed of two lognormally distributed peaks were studied. The distributions differed with regard to the variance of the peaks, which were designed to investigate a range of distributions containing only discrete, only smooth or both features in the same distribution. Three different signal-to-noise ratios were studied: 2000, 200 and 20. A new metric is proposed to compare the distributions reconstructed from the different regularization methods with the true distributions. The metric is designed to penalise reconstructed distributions which show artefact peaks. Based on this metric, MTGV regularization performs better than Tikhonov and L1 regularization in all cases except when the distribution is known to only comprise of discrete peaks, in which case L1 regularization is slightly more accurate than MTGV regularization.

Description of artefacts in the PowerPlex Y23® system associated with excessive quantities of background female DNA

Male on female sexual assault cases that involve azoospermic individuals, those where the male has penetrated but failed to ejaculate, those where there has been an extended interval between the sexual assault and sample collection or where there has been only digital penetration are often difficult to investigate by employing traditional autosomal STR testing. Such cases often involve minimal amounts of male DNA either being deposited initially or remaining after the passage of time. These cases are often further complicated by the presence of large amounts of female DNA compared to the relatively small amounts of male DNA on the intimate samples taken. Y-STR kits provide a solution that allows targeting of male DNA in a mixed male/female sample. However, large quantities of excess female DNA have the potential to generate non-specific artefact peaks. Here we characterise a number of previously reported artefacts observed in the PowerPlex® Y23 system. We demonstrate that some of these artefacts can impact on profile interpretation and that they are highly dependent on the levels of female DNA present. These artefacts have been characterised to assist practitioners with the interpretation of such samples.

Performance of the PowerPlexY23® kit on trace samples in forensic genetic casework

Y-chromosomal short tandem repeats (Y-STRs) can be analysed with the AmpFlSTR®Yfiler® kit (Yfiler, Life Technologies (LT)) from trace samples in forensic case-work. However, the results are not reliable when the ratio of female:male DNA is high. The aim of this study was to investigate the performance of the PowerPlex®Y23 kit (PPY23, Promega) on trace samples from crime scenes. A total of 100–200pg male DNA was analysed with the PPY23 kit from 45 trace samples. Amplification of Y-STRs with the PPY23 kit was successful for 34 (76%) of the trace samples. Reproducible artefact peaks in the reading area (80–450 base pairs) were found in up to 29% of the trace samples typed with the PPY23 kit. Significantly higher female:male DNA ratios were observed in profiles with artefact peaks compared to those of profiles with no artefact peak. It was concluded that the quality of Y-STR analyses of trace samples would not be improved by replacing the Yfiler kit with the PPY23 kit in our laboratory.

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10–12h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2–3h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpFℓSTR® SGM Plus™ (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion® Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO® thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a “DNA-6h” service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution.

Perturbation of ion trajectories by resonant excitation leads to occurrence of ghost peaks

AbstractWe have shown in a recent paper (Kocher F, Favre A, Gonnet F, Tabet JC J. Mass Spectrom. 1998; 33: 921) that under particular ion trapping conditions, i.e. space charge, artefacts called ghost peaks were displayed in mass spectra and isolation spectra. All experimental factors which modified the space‐charge strength, influenced the intensities of the ghost peaks. It was demonstrated that only the injected ions having qz ≤0.25 (βz ≤ 2/11) at the beginning of the analytical scan step gave rise to the formation of such artefact peaks. In fact, the ion cloud defocused by space charge was subjected to high‐order multipole fields (e.g. 22‐pole, Wang Y, Schubert M, Franzen J, Proc. 44th ASMS Conf. Mass Spectrom. and Allied Topics, Portland OR, 1996; p. 131), so that the trajectories of the analysed ions were destabilized thus affecting the ion ejection which then occurred at qz = 0.908 (βz = 1, natural boundary). However, ions having 0.25 (βz ≤ 2/11) ≤qz ≤0.45 (βz ≤ 1/3), at the beginning of the analytical scan step, were not destabilized, and were correctly ejected where the axial modulation was applied (βz ≤ 1/3). Thus, under extreme space‐charge conditions, the ion excursion increased, the influence of the non‐linear resonance at βz = 2/11 was enhanced, ion excursion was more enhanced, and the ions were not ejected at βz = 1/3 but at βz = 1.We shown here that, again under the influence of the non‐linear resonance at βz = 2/11, under space‐charge conditions the ion excursion increased, and the application of a resonant excitation dramatically enhanced this ion excursion, compared to only the isolation step as was performed in the previous work. This led to these ions not being ejected at βz = 1/3 but rather at βz = 1, and thus being displayed as ghost peaks in the collisionally induced dissociation (CID) spectra. This behavior could take place for other working ejection points, which occur at non‐linear resonances (e.g. βz = 2/11 as well as βz = 2/3). Copyright © 2001 John Wiley & Sons, Ltd.

Improved purity assessment of high-performance liquid chromatography diode array detection data for overcoming the presence of the non-linearity artefact

Orthogonal projection approach (OPA) and needle search (NS) have been applied to assess the purity of peaks in a spectrochromatogram recorded with a high-performance liquid chromatograph coupled with a diode array detector. The background disturbances result in a non-linearity artefact at low concentrations and give an additional variation in the data set, which is difficult to correct due to the small magnitude of the baseline anomalies. The presence of non-linearities leads to artefact peaks in both OPA and NS plots. Decision criteria and certain modifications were introduced in OPA and NS in order to obtain more informative results and to distinguish artefact peaks from peaks corresponding to minor components. Small positive offsets were included in NS and a threshold curve obtained from the standard of the main compound was introduced in OPA. Both methods perform equally well for the detection of the impurities in the presence of non-linearities. Although, OPA requires a certified standard and additional experimental work.

Study of ghost peaks resulting from space charge and non‐linear fields in an ion trap mass spectrometer

Ion trap improvements require some geometrical modifications which can alter significantly the relative linear and non-linear field contributions. In this work, where the ion trap was interfaced with an electrospray source, it was shown that under particular ion trapping conditions due to space charge, artefacts called ghost peaks are displayed in the mass spectra. They are characterized by particular width and shape and appear at twice the m/z ratio of the expected peaks (for positive and negative ions) when the analyzed ions are ejected at βz=1/3 (extended mass range mode). They were unambiguously identified by using isolation sequences. The various experimental factors governing the observation of ghost peaks were explored in order to rationalize their origin. Especially the capillary exit voltage, ion accumulation time and low m/z cut-off of the analytical scan, which modify the space charge strength, influence the ghost peak intensities. Furthermore, it is evidenced that only the injected ions which are located at qz⩽0.25 (βz⩽2/11) at the beginning of the analytical scan step give rise to the formation of such artefact peaks. Under these experimental conditions, the ion cloud defocused by space charge is subjected to high-order multipole fields (e.g. 22-pole). Then the motion of the analyzed ions is destabilized, affecting the ion ejection. It seems that two ion populations can co-exist: (i) one submitted to a normal ion ejection at qz=0.45, where the axial modulation is applied, i.e. normal peaks, and (ii) a second ejected at the natural qz=0.908 boundary, leading to ghost peaks at twice the m/z value of normal signals. © 1998 John Wiley & Sons, Ltd.

Study of ghost peaks resulting from space charge and non-linear fields in an ion trap mass spectrometer

Ion trap improvements require some geometrical modifications which can alter significantly the relative linear and non-linear field contributions. In this work, where the ion trap was interfaced with an electrospray source, it was shown that under particular ion trapping conditions due to space charge, artefacts called ghost peaks are displayed in the mass spectra. They are characterized by particular width and shape and appear at twice the m/z ratio of the expected peaks (for positive and negative ions) when the analyzed ions are ejected at βz=1/3 (extended mass range mode). They were unambiguously identified by using isolation sequences. The various experimental factors governing the observation of ghost peaks were explored in order to rationalize their origin. Especially the capillary exit voltage, ion accumulation time and low m/z cut-off of the analytical scan, which modify the space charge strength, influence the ghost peak intensities. Furthermore, it is evidenced that only the injected ions which are located at qz⩽0.25 (βz⩽2/11) at the beginning of the analytical scan step give rise to the formation of such artefact peaks. Under these experimental conditions, the ion cloud defocused by space charge is subjected to high-order multipole fields (e.g. 22-pole). Then the motion of the analyzed ions is destabilized, affecting the ion ejection. It seems that two ion populations can co-exist: (i) one submitted to a normal ion ejection at qz=0.45, where the axial modulation is applied, i.e. normal peaks, and (ii) a second ejected at the natural qz=0.908 boundary, leading to ghost peaks at twice the m/z value of normal signals. © 1998 John Wiley & Sons, Ltd.

Isolation of dinophysistoxin-2 and the high-performance liquid chromatographic analysis of diarrhetic shellfish toxins using derivatisation with 1-bromoacetylpyrene

The rare diarrhetic shellfish toxin, dinophysistoxin-2 (DTX-2), was isolated from the digestive glands of mussels ( Mytilus edulis). This was achieved by chromatography on silica and Sephadex LH-20 followed by reversed-phase solid phase extraction and semi-preparative high-performance liquid chromatography (HPLC) with an Ultremex C 18 column. Using 1-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, the diarrhetic shellfish toxins, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and DTX-2, were determined by HPLC with fluorimetric detection. Derivatisation using BAP was compared with 9-anthryldiazomethane (ADAM) and, although the latter exhibited a four-fold better sensitivity, the BAP method gave fewer artefact peaks from reagent decomposition. The limits of detection of OA and DTX-2 were 0.4 ng on-column using BAP, which permits this method to be used for the regulatory control of these toxins in shellfish.

Graphical method for artefact peak interpretation, and methods for their rejection, using double and triple sector magnetic mass spectrometers

AbstractThe problem of artefact peaks, in spectra obtained from various tandem mass spectrometric scans on instruments of both forward and reverse geometry, is examined. The filtering conditions imposed by additional analyzers are investigated, in order to evaluate the potential of several different triple sector geometries to reduce specific interferences. No previous evaluation of the rejection ability for artefacts of different origins has been presented with respect to the various geometries. In order to gain a better insight into the origin of various artefacts, and to more easily explain their occurrence in certain geometries, a new graphical representation was developed in order to take into account the effects of three analyzer fields. This approach allows different linked scans to be projected within a cube, while also qualitatively proposing an explanation for the presence or absence of artefact peaks. New scans can also be conceived by means of this method, as is the case with a new selected mass ion kinetic energy (SMIKE) scan, which allows artefacts present in MIKE scans performed using reverse geometry instruments to be determined.

Determination of artefact peaks in mass‐analysed ion kinetic energy spectra using a new linked scan for ebe geometry magnetic sector instruments

AbstractThe possible presence of artefact peaks in mass‐analyzed ion kinetic energy (MIKE) spectra has been recognized when such scans are performed using a two‐sector instrument of reverse (BE) configuration. Since the presence of an artefact peak can be misleading when interpreting a spectrum, a new scan is proposed which allows these artefacts to be identified. This scan involves linking and scanning the two electrostatic sectors of an E1BE2 geometry instrument, while maintaining the magnet at the mass selected for the MIKE scan. This selected mass‐analyzed ion kinetic energy (SMIKE) scan at E1=E2 results in a spectrum containing only artefact peaks that can be present in the corresponding MIKE spectrum.

Artefact peaks in particle size distributions measured by the electrical sensing zone (coulter counter) method

Artefact peaks on the coarse side of particle size distributions measured via the electrical sensing zone (ESZ) technique for a series of copolymer latices have been examined. These artefacts result from particles entering the orifice mouth at the periphery. It is shown that the ‘volume’ shift of the artefact peak relative to the main or normal peak may be calculated from the equation ▪ where dφ/ dx is the axial component of the potential gradient across the mouth of the orifice, of radius R, at a radial distance r from the axis; α′ and β′ are constants with values of 0.0324 and 1.082 respectively. The relative modal heights of the artefact and main peaks are also accounted for. The occurrence of artefact peaks can introduce difficulties in the use of the ‘half-count’ method of calibration; a preferred alternative method involves identification of the modal value of the main peak in the measured ESZ size distribution of the reference material used for calibration.

The observation of metastable transitions in a double-focussing mass spectrometer using a linked scan of the electric sector and magnetic sector fields

A detailed study has been made of the observation of metastable transitions in the first field-free region of a double-focussing mass spectrometer of Nier—Johnson geometry by scanning simultaneously the magnetic field B and the electric sector field E such that B/ E remains constant throughout the scan. The B/ E linked scan provides a convenient method of determining the daughter ions formed from a chosen parent ion and, because of the transmission characteristics associated with this type of scan, sharp peaks are observed irrespective of the amount of internal energy released as translational energy of the fragments. This greatly facilitates the accuracy with which the mass ratio of parent and daughter ion can be determined and also allows a wide range of mass ratios to be investigated at full source sensitivity. The scan gives equally good results when used to study collision-induced decompositions. The origin of several low-intensity artefact peaks is established and methods of recognising such peaks are discussed. The B/ E linked scan complements other methods of investigating metastable transitions already in use and is likely to find most application in the study of analytical problems.

Gas-Chromatographic Separation of Inorganic Chlorides and its Application to Metal Analysis: I. Qualitative and Quantitative Aspects of the Gas-Chromatographic Separation Procedure

Abstract A study has been made of the potentialities of gas chromatography as a tool for the analysis of metals and alloys. As is demonstrated in the present paper, gas chromatography can be used for the separation and the quantitative determination of inorganic chlorides. With proper adaptation of the apparatus and technique to the reactivity of these compounds, satisfactory chromatograms were obtained, showing good peak symmetry, stable baselines, and absence of artefact peaks. Of nineteen substances studied, having boiling points between 50 and 250°C, all but a few were determined accurately in mixtures. The exceptions are the very unstable vanadium tetrachloride and antimony pentachloride (dissociating readily even at relatively low temperatures) as well as an oxychloride of molybdenum, of which the identity was uncertain. A number of determinations carried out on a mixture of the tetrachlorides of silicon and tin under various conditions showed a good reproducibility: standard deviations of 0.2 to 0....