Identification Points System Research Articles

A global inter-laboratory study to assess acquisition modes for multi-compound confirmatory analysis of veterinary drugs using liquid chromatography coupled to triple quadrupole, time of flight and orbitrap mass spectrometry

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of “identification points”. A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors.In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points.

The assessment of selectivity in different Quadrupole-Orbitrap mass spectrometry acquisition modes.

Selectivity of the confirmation of identity in liquid chromatography (tandem) mass spectrometry using Q-Orbitrap instrumentation was assessed using different acquisition modes based on a representative experimental data set constructed from 108 samples, including six different matrix extracts and containing over 100 analytes each. Single stage full scan, all ion fragmentation, and product ion scanning were applied. By generating reconstructed ion chromatograms using unit mass window in targeted MS(2), selected reaction monitoring (SRM), regularly applied using triple-quadrupole instruments, was mimicked. This facilitated the comparison of single stage full scan, all ion fragmentation, (mimicked) SRM, and product ion scanning applying a mass window down to 1 ppm. Single factor Analysis of Variance was carried out on the variance (s(2)) of the mass error to determine which factors and interactions are significant parameters with respect to selectivity. We conclude that selectivity is related to the target compound (mainly the mass defect), the matrix, sample clean-up, concentration, and mass resolution. Selectivity of the different instrumental configurations was quantified by counting the number of interfering peaks observed in the chromatograms. We conclude that precursor ion selection significantly contributes to selectivity: monitoring of a single product ion at high mass accuracy with a 1 Da precursor ion window proved to be equally selective or better to monitoring two transition products in mimicked SRM. In contrast, monitoring a single fragment in all ion fragmentation mode results in significantly lower selectivity versus mimicked SRM. After a thorough inter-laboratory evaluation study, the results of this study can be used for a critical reassessment of the current identification points system and contribute to the next generation of evidence-based and robust performance criteria in residue analysis and sports doping.

Rapid, high performance method for the determination of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K1) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K2 family and metabolite of K1 in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K1, K1 2,3-epoxide (K1O), and MK-4 in human serum and plasma at concentrations in the ng/L–μg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal “endogenous suppressors” and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760μg/L. MK-4 was not detected in either of the two groups.

Development and validation of highly selective screening and confirmatory methods for the qualitative forensic analysis of organic explosive compounds with high performance liquid chromatography coupled with (photodiode array and) LTQ ion trap/Orbitrap mass spectrometric detections (HPLC-(PDA)-LTQOrbitrap)

An LTQ-Orbitrap FTMS is a new (hybrid) mass spectrometric (MS) analyzer. It allows for the acquisition of full scan MS(n) (n-stage fragmentations, n=1-n) spectra with the linear ion trap detector (LTQ) at high speed and/or with the Fourier Transform-detector (Orbitrap) with ultra high mass resolution (>60,000 at m/z<400amu) and high mass accuracy (≤1ppm with internal calibration). In addition it may be coupled with liquid chromatography (LC) with photo diode array (PDA) detection. Two methods for the forensic screening and confirmation of all common trace explosives in post-blast residues have been developed on this instrument using atmospheric pressure chemical ionization (APCI). In one run, the nitrogen-containing explosives are analyzed with the combination of "LC-(PDA)-APCI(-)-LTQ MS(2)/Orbitrap FTMS" (Method 1). In another run, peroxide explosives are analyzed with "LC-APCI(+)-LTQ MS(2)/Orbitrap FTMS" (Method 2). The performance of both methods has been validated according to procedures defined in the EU COMMISSION DECISION implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (DC 2002/657/EC) and other standards (NEN 17025 and NEN 7777). The methods are highly selective due to the simultaneous utilization of the Orbitrap FTMS and LTQ MS(2), both of which are highly selective detectors Tested explosive compounds can be detected in the molecular ion form by the Orbitrap analyzer with minimal mass interference in different matrices when using an extremely narrow mass tolerance detection window (≤2ppm). The identification of a detected compound follows an identification point system. Experimental results show that almost all explosive compounds meet the confirmation criteria (minimum 4 points) required for the positive identification by the DC 2002/657/EC.

Rapid and sensitive determination of Sudan dyes in hot chilli products by solid-phase extraction directly combined with time-of-flight mass spectrometry

A novel method based on combination of a solid-phase extraction (SPE) with humic acid-bonded silica (HAS) as sorbent with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS) was developed for rapid and sensitive determination of four Sudan dyes in hot chilli products. A SPE procedure was optimized for the improvement of extraction efficiency and the removal of the matrix interferences such as the pigments and lipophilic compounds from hot chilli products. Under the optimum conditions, the desorbed analytes from the SPE sorbent allowed direct analysis by ESI-QTOF MS without time-consuming chromatographic separation. In the positive ion mode and MS/MS mode, linearities for concentration ranges of two orders of magnitude were obtained with the correlation coefficients (R2) greater than 0.9990; the limit of detection (LOD) and limit of quantification (LOQ) for four Sudan dyes were in the ranges of 1.1–4.3 and 3.8–14.3 ng g−1 depending on matrices, respectively. The recoveries of four Sudan dyes in two matrices ranged from 85.6 to 119.7%, with relative standard deviations (n = 4, 3) less than 11.3% and 11.6%. The developed method was successfully applied for the determination of Sudan dyes in six hot chilli products and the positive samples were confirmed with a high number of identification points (IPs) according to IPs system defined by Commission Decision 2002/657/EC.

Investigation of the identification point system adaptation in cocaine, benzoylecgonine and ecgonine methyl ester using a single quadrupole mass spectrometer

At present, no official criteria exist for drug identification using single quadrupole mass spectrometers although the European Union (EU) criteria for compound identification have been adopted. These criteria are evaluated with respect to the confirmation of cocaine and its metabolites by single quadrupole liquid chromatography/mass spectrometry (LC/MS) and problems are highlighted. Spiked samples, proficiency testing samples, certified reference materials and samples from real cases that had screened positive for cocaine derivatives by immunoassay were subjected to confirmation by LC/MS using single ion monitoring with in-source fragmentation. The EU criteria for compound identification were applied for the confirmation of cocaine, benzoylecgonine and ecgonine methyl ester. The use of the identification point (IP) system in spiked, proficiency testing samples and certified reference materials provided acceptable results in all cases while in some cases real positive samples did not provide acceptable results. Failure to meet the EU criteria was attributed to low fragmentation at the lower concentrations and the ion suppression effect while both factors affected compliance with the IP system. The identification of cocaine and its metabolites was considerably improved by using a combination of ammonium formate and formic acid as the LC mobile phase. It appears that poor in-source fragmentation in single quadrupole LC/MS and ion suppression may constitute a problem with drug identification when implementing the IP system in real samples, resulting in false negative results. Further investigation is needed for the use of such IP systems to be suitable for use in LC/MS methods.

Evaluating polymer monolith in-tube solid-phase microextraction coupled to liquid chromatography/quadrupole time-of-flight mass spectrometry for reliable quantification and confirmation of quinolone antibacterials in edible animal food

A simple, rapid, and sensitive method is presented to determine seven trace quinolone antibacterials simultaneously in milk, egg, chicken and fish. This method is based on the combination of polymer monolith in-tube solid-phase microextraction with liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS). LC/ESI-QTOF-MS offers the capability of unequivocal identification of target compounds from complex matrices, as well as the possibility of quantitation at low-level concentrations in real samples. The extraction was performed with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic column. Under the optimized extraction conditions, good extraction efficiencies for the targets were obtained with no matrix interference in the subsequent LC/ESI-QTOF-MS. Good linearities were obtained for seven quinolones with the correlation coefficients ( R) above 0.9951. The limits of detection ( S/ N = 3) for seven quinolones were found to be 0.3–1.2 ng/g in egg, 0.2–3.0 ng/mL in milk, 0.2–0.7 ng/g in chicken and 0.2–1.0 ng/g in fish. The recoveries of quinolones spiked in four different matrices ranged from 80.2 to 115.0%, with relative standard deviations less than 14.5%. The developed method was applied for the determination of quinolone residues in animal-producing food, and the positive samples were confirmed with high number of identification points (IPs) according to the IP system defined by the European Union (Commission Decision 2002/657/EC).

False‐positive liquid chromatography/tandem mass spectrometric confirmation of sebuthylazine residues using the identification points system according to EU directive 2002/657/EC due to a biogenic insecticide in tarragon

In pesticide residue analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) the confirmation of a sebuthylazine finding in a tarragon (Artemisia dranunculus) sample was demonstrated to be false positive. A coeluting interfering matrix compound produced product ions in MS/MS analysis, perfectly corresponding to the multiple reaction monitoring (MRM) of two sebuthylazine transitions. Using the EU directive 2002/657/EC which regulates the confirmation of suspected positive findings would have resulted in a false-positive finding. A third LC/MS/MS transition with a deviant ion ratio and a gas chromatography (GC)/MS/MS analysis revealed the false-positive results. With optimized high resolving ultra-performance liquid chromatography (UPLC) conditions it was possible to separate spiked sebuthylazine from the interfering matrix compound. Using its exact mass and isotope ratios from LC/time-of-flight (TOF) MS measurements, the compound was identified as nepellitorine, a - not surprising - endogenous alkamide in tarragon (Arthemisia dranunculus). False-positive results, especially in heavy matrix samples such as herbs, can be dealt with by further confirmatory analysis, e.g. a third transition, GC analysis if possible or more advantageous by an orthogonal criterion like exact mass.

Application of gas chromatography/tandem quadrupole mass spectrometry to the multi-residue analysis of pesticides in green leafy vegetables.

A new, sensitive and specific method has been developed for the simultaneous determination of 129 pesticides in lettuce and other green leafy vegetables. The samples were extracted with acetonitrile and co-extractives such as fatty acids and pigments were removed using dispersive solid-phase extraction (dispersive-SPE) with primary secondary amine (PSA) and graphitized carbon black (GCB). All pesticides were analyzed in a single injection gas chromatography/tandem quadrupole mass spectrometry (GC/MS/MS) acquisition method. Two multiple reaction monitoring (MRM) transitions of precursor ions fragmenting into product ions were recorded for the targeted pesticides, thus fulfilling the EU identification points system criteria for the identification of contaminants (2002/657/EC). Calibration curves were determined using matrix-matched standards, and exhibited excellent linearity at two orders of magnitude from 0.005 to 0.5 mg/kg for almost all the pesticides studied (R(2) > or = 0.99). The analytical performance was demonstrated by the analysis of lettuce samples spiked at five concentration levels ranging from 0.005 to 0.5 mg/kg for each pesticide. The recovery and repeatability results satisfied SANCO/2007/3131 criteria (i.e. average recoveries were in the range 70-120% with RSDs < or =20%) for 114 of the 129 pesticides at the 0.005 mg/kg spiking level, and for almost all pesticides at the higher spiking levels. The methodology was applied successfully to identify and quantify pesticide residues in leafy vegetable samples such as lettuce, cabbage and leek.

Identification and confirmation of chemical residues in food by chromatography-mass spectrometry and other techniques

A quantitative answer cannot exist in analysis without a qualitative component to give enough confidence that the result meets the analytical needs (i.e. the result relates to the analyte and not something else). Just as a quantitative method must typically undergo an empirical validation process to demonstrate that it is fit for purpose, qualitative methods should also empirically demonstrate that they are suitable to meet the analytical needs. However, thorough qualitative method validation requires analysis of a great number of samples (possibly more than can be reasonably done), which is generally avoided due to the time and the effort involved. Instead, mass spectrometry (MS) is generally assumed to be the gold standard for qualitative methods, and its results are typically unquestioned. For example, a system was developed by European regulators of veterinary drug residues in food animals (2002/657/EC), in which the number of identification points given in MS analyses depends on the general degree of selectivity of the MS technique used. This well-defined approach gives a definite answer for decision-makers, so it has grown in popularity. However, the identification-points system is not scientific. The reality is that each situation requires information gathering and careful deductive thinking on the part of the analyst to make MS identifications. Rather than devise arbitrary requirements that need to be met by an unthinking analyst, we remind the analytical community that confirmation can be given only if two or more independent analyses are in agreement, preferably using orthogonally selective (independent) chemical mechanisms. In this article, we discuss the proper use of terminology, highlight the identification power of various MS techniques, demonstrate how MS identifications can fail if precautions are not taken, and re-assert the value of basic confirmation practices, qualitative method validation, information checklists, routine quality-control procedures, and blind proficiency-test analyses.

Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC–TOF): A novel tool for multiresidue screening of veterinary drugs in urine

A multiresidue method for the screening of veterinary drugs in urine with ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF) is proposed. The method covers currently more than 100 analytes belonging to different families of veterinary drugs. Urine samples are simply diluted and injected unfiltered into the UPLC–TOF. The short run time permit high-throughput of large series under a routine environment.The suggested approach includes the detection of some metabolites which are relevant to the urine matrix. The paper discusses the usefulness of metabolites as additional confirmation criteria for positive findings and suggests a revisiting of the identification point system in the light of additional resolution, as provided by the TOF and the UPLC technology. The proposed multiresidue approach was further broadened by monitoring drug group specific collision induced fragments (CID). Examples show the monitoring of generic CID fragments typical for sulfonamides and penicillines.