A component tracking algorithm for accelerated and improved liquid chromatography-mass spectrometry method development

A method for tracking of sample components during liquid chromatography-mass spectrometry (LC-MS) method development has been proposed. The method manages to, fully automatically and without user intervention, find the chromatographic peaks in the data sets, discriminate them to sample components and track them when the separation conditions have been changed. The algorithm utilises the resolution obtained from all considered data sets and has the ability to discriminate the non informative parts. The technique has a great sensitivity even in cases where a majority of the tracked components cannot easily be spotted by means of traditional total ion chromatogram (TIC) or base peak chromatogram (BPC) representations. The method was tested on an experimental sample using six different columns and an average of 79% of the suggested sample components could be successfully tracked at a minimum area of 0.05% of the main component in the sample. 66 components with 79-92% of the total suggested component area were able to be tracked between all data sets. The method could be used to rapidly investigate selectivity during different types of separation conditions.     

High-pass filters for spectral background suppression in airborne passive Fourier transform infrared spectrometry

High-pass (HP) digital filtering and second-derivative (SD) filtering are evaluated as methods of removing background contributions from spectra collected by passive Fourier transform infrared spectrometry. In measurements performed with a downward-looking spectrometer mounted on an aircraft platform, the effects of non-constant background radiance from the ground make it challenging to build automated classifiers for detecting an analyte of interest. Applying HP digital filtering to the spectra to remove background contributions is evaluated as a strategy to help improve classifier performance. This methodology is tested by building classifiers for detecting heated ethanol plumes released from a portable emission stack. The classifiers are trained with data collected on the ground with the spectrometer viewing the plumes against a synthetic backdrop designed to simulate a terrestrial radiance source. The resulting classifiers are tested with data collected by the same spectrometer mounted on an aircraft flying over the emission stack. Support vector machines are employed as a classification algorithm with HP filtered spectra used as input patterns. Butterworth filters are used to implement HP digital filtering, while Savitzky-Golay filters are used to implement SD filtering. Significant improvement in classification performance is achieved by use of the HP filters. Because of variation in backgrounds between the training and prediction data, the best classifier obtained with unfiltered spectra is unable to detect ethanol in 37% of the test cases. HP filtering of spectra with an optimized Butterworth filter (order 8, cutoff frequency 0.060) improves the prediction results, resulting in no missed ethanol detections and false positive rates of less than 0.4%.     

Detecting and aligning peaks in mass spectrometry data with applications to MALDI

In this paper, we address the peak detection and alignment problem in the analysis of mass spectrometry data. To deal with the peak redundancy problem existing in the MALDI data acquired in the reflectron mode, we propose to use the amplitude modulation technique in peak detection. The alignment of two peak sets is formulated as a non-rigid registration problem and is solved using a robust point matching (RPM) approach. To align multiple peak sets, we first use a super set method to find a common peak set among all peak sets as a standard and then align all peak sets to the standard using the robust point matching approach in a sequential manner (i.e. We align only one peak set to the standard each time, thus reducing the multiple peak set alignment problem to a simpler two peak set alignment problem). Experimental results from a study of ovarian cancer data set show that the quantitative cross-correlation coefficients among technical replicates are increased after peak alignment. Additional comparisons also demonstrate that our method has a similar performance as the hierarchical clustering method, although the implementations of these methods are different.     

Gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry in quantifying fatty acids

This critical overview covers current analytical methods and future developments in quantitative determination of fatty acids (FAs), emphasizing sample extraction, derivatization and instrumental analysis with gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS²). We compare the benefits and the drawbacks of these two analytical techniques.We consider the well-established GC/MS method with pre-derivatization to be a traditional technique in terms of highly standardized sample-preparation procedures, affordability and readily available library searching for compound identification. However, the complicated derivatization steps required prior to instrumental analysis with GC/MS take a long time, with loss and transformation of FAs, low recovery and poor reproducibility.HPLC/MS² without derivatization shows the benefits of simple, mild sample-processing conditions, satisfactory recovery, short running time and high selectivity and sensitivity, which may allow it to become a viable alternative to GC/MS for the analysis of FAs in the years ahead.     

Multi-location peak parking method: an important new tool for the study of mass transfer kinetics in liquid chromatography

The peak parking (PP) method probes the longitudinal diffusion coefficient of a compound at a single location along the chromatographic column. We extended to a so-called multi-location peak parking (MLPP) method, in which a large number of axial locations along the column are selected in order to check the validity of the conventional PP method and to reveal possible defaults in the structure of the packed bed or pitfalls of the PP and the MLPP methods. MLPP was applied to a series of HILIC columns, including a 5.0 μm Venusil, a 3.0 μm Luna-diol, three 2.7 μm Halo, and a 1.7 μm Kinetex columns. The results demonstrate that the MLPP method may reveal local heterogeneities in the axial diffusion of small retained low molecular weight compounds along the column. Most importantly, experiments show that the sample zone should not be parked in the entrance of the column (i.e., at <1/10 th of the column length). The abrupt drop in the flow rate considerably affects the peak shape and prevents scientists from using the conventional PP method. Practical solutions to cope with that problem are proposed and their success/failure are discussed.     

液相色谱-质谱联用在兴奋剂检测中的应用及进展

液相色谱-质谱联用技术已越来越广泛地应用在兴奋剂的检测中,其中包括对各类小分子兴奋剂和肽类激素等的检测。本文综述了近年来液相色谱-质谱联用在兴奋剂检测中的筛选、确证和定量方面的应用及进展情况,并讨论了相关的检测标准。     

Confirmatory and quantitative analysis using experimental design for the extraction and liquid chromatography-UV, liquid chromatography-mass spectrometry and liquid chromatography-mass spectrometry/mass spectrometry determination of quinolones in turkey muscle

The aim of this work is to established methods for determination of quinolones (ciprofloxacin, danofloxacin, enrofloxacin, difloxacin and flumequine), regulated by European Union, and sarafloxacin in turkey muscle. An experimental design has been applied for the optimization of the factors that influence the extraction of quinolones from turkey muscle in order to determine the experimental conditions for their extraction with high recoveries. Liquid chromatography with ultraviolet detection (LC-UV), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) have been used for the simultaneous quantification of quinolones antibiotics in turkey muscle. The proposed methods have been validated according to the Food Drugs Administration guideline and presents the limit of quantification below the maximum residue limits established by the European Union for quinolones in turkey muscle. The methods developed have been applied to quantification of enrofloxacin and its main metabolite ciprofloxacin in samples of turkey muscle obtained from animals treated with enrofloxacin.     

Algorithms for the assessment of peak purity in liquid chromatography with photodiode-array detection. Part II

Two modifications of the algorithm based on the Gram-Schmidt orthogonalization technique for the assessment of peak purity are presented. The performance of this approah is investigated for liquid chromatography with photodiode-array detection (LC-DAD) data, although its applicability is not restricted to this experimental model. This method is applied to simulated and experimental data where two compounds are eluting, but can be applied when more compounds are eluting. The results are compared with the ones obtained previously with the first version of this algorithm.     

Simultaneous determination of five flavonoids in licorice using pressurized liquid extraction and capillary electrochromatography coupled with peak suppression diode array detection

Pressurized liquid extraction (PLE) and capillary electrochromatography (CEC) methods were developed for the simultaneous determination of five flavonoids, namely liquiritin, isoliquiritin, ononin, liquiritigenin and isoliquiritigenin, in licorice using baicalein as internal standard (IS). Peak suppression technique was used for the quantification of ononin because of its poor resolution with isoliquiritin. The analysis was performed on a Hypersil C₁₈ capillary (3 μm, 100 μm/25 cm) with a mixture of 10 mM phosphate buffer (pH 3.0)/ACN (65:35, v/v) as mobile phase running at 25 kV and 30 °C. The detection wavelengths were set at 275 nm (without reference wavelength for liquiritin and liquiritigenin), 360 nm (without reference wavelength for isoliquiritin and isoliquiritigenin) and 254 nm (with reference wavelength of 405 nm for ononin). All calibration curves showed good linearity (R² > 0.9993) within the test ranges. The LOD and LOQ were lower than 2.1 and 8.3 μg/mL, respectively. The RSDs of intra- and interday for relative peak areas of five analytes to IS were less than 3.8 and 4.7%, respectively, and the recoveries were 98.2–103.8%. The validated method was successfully applied to the quantitative analysis of five flavonoids in licorice, which is helpful to its quality control.     

超高效液相色谱-高分辨质谱联用结合整合过滤策略全面分析茶树花中化学成分

茶树花与茶鲜叶同为茶树的生物产出,但茶树花往往被视为茶叶生产过程中的废物被舍弃,造成了茶树花资源的极大浪费.目前对于茶树花中化学成分的分析主要集中在氨基酸、茶多酚等单一类型化学成分上,对于茶树花中多类化学成分的同时分析仍鲜见报道.研究者对于茶树花中所含化学成分的种类和含量不完全清楚,成为制约茶树花深度开发与利用的重要原...     

Optimisation of data handling and detection conditions for automated chromatographic assay software

Summary The principle of automated chromatographic peak detection and analysis software is summarized, and critical steps are systematically studied. As the only parameter to be entered is the acquisition frequency, evaluation of its effect on software performance is discussed. In the case of relatively noisy chromatograms, it is shown experimentally that numerous points per peak have to be taken, leading to quite fast computer acquisition procedures. The use of discrete Fourier transform filtration techniques can modify peak shapes and a comparative study evaluates the relative errors induced in the shapes and characteristics of the chromatographic profiles. Optimisation of filtering conditions is achieved and it is shown that for a filter position only 2% of the Nyquist frequency no deformation occurs in the chromatographic profile. Detection of the start and finish of chromatographic peaks is optimized according to a simple four step iterative procedure. In the case of simulations, the difference between the values used to simulate peaks and those calculated by the software are less than 1%.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

Development of analytical methods for multiresidue determination of quinolones in pig muscle samples by liquid chromatography with ultraviolet detection, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry

This paper presents a comparison between liquid chromatography with ultraviolet detection (LC-UV), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods developed for the multiresidue determination of 8 quinolones, around their maximum residue levels (MRLs) in pig muscle. The procedure involves common extraction of the quinolones from the tissues by traditional extraction, a step for clean-up and preconcentration of the analytes by solid-phase extraction (SPE) and a subsequent liquid chromatographic analysis. The methods present satisfactory results of linearity, precision and limits of quantification much lower than the MRLs established by the European Union for quinolones in pig tissues.     

Evaluation of the interaction of bioactive compounds in Cortex Pseudolarix and Radix Stephaniae by the microdialysis probe coupled with high performance liquid chromatography-mass spectrometry

An efficient and convenient method, biological fingerprinting chromatogram analysis is presented, which is applied to the comparison of fingerprinting chromatograms of the extracts of Chinese herbal medicines after the interaction with biological systems (cell, DNA, protein, etc.). The method was established for the purpose of screening and analysis of the multiple bioactive compounds in herbal medicines. In this work, microdialysis sampling combined with high performance liquid chromatography-mass spectrometry (HPLC-MS) was studied for binding property of MCF-7 and multidrug resistant MCF-7 cell systems. The results showed that pseudolaric acid A (PAA) and pseudolaric acid B (PAB) in the cortex of Pseudolarix kaempferi (Lamb.) Gorden can easily bind to the MCF-7 cells ranging from 0 to 16.3% (PAA) and from 0 to 35.7% (PAB), and another compound, tetrandrine (TET) from the root of Stephania tetrandrae S. Moore, showed higher binding activity with multidrug resistant MCF-7 cells ranging from 0 to 39.9%.     

An improved method for analyzing chlormequat and mepiquat in source waters by solid-phase extraction and liquid chromatography-mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of chlormequat (CQ) and mepiquat (MQ) in source waters with high sensitivity and specificity was established using WCX cartridges (150 mg/6 mL) for pre-concentration of the samples and using the CAPCELL PAK CR 1:4 (2.0 mm × 150 mm 5 μm, SCX:C18 = 1:4) column containing strong cationic exchange resins and C18 for separation. The method could solve the problem for pre-concentrating ionic compounds from water samples and avoid the MS instrument fouling problem accompanied with the use of ion-pair reagents. After the optimization of analytical conditions, quantification was achieved in the positive electrospray ionization mode using selected ion monitoring. The samples were analyzed with multi-channel mode with quantification performed at 30 V cone voltage to ascertain the sensitivity and qualitative analysis performed at 60 V cone voltage simultaneously. The method detection limits (MDLs) of CQ and MQ in source water were determined to be 14 and 22 ng L⁻¹. Finally, the method was used to quantify CQ and MQ in several source waters, which gave a level ranging from below the quantitation limit to 28 ng L⁻¹.     

Peak clustering in two-dimensional gas chromatography with mass spectrometric detection based on theoretical calculation of two-dimensional peak shapes: the 2DAid approach

A method is presented to facilitate the non-target analysis of data obtained in temperature-programmed comprehensive two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToF-MS). One main difficulty of GC×GC data analysis is that each peak is usually modulated several times and therefore appears as a series of peaks (or peaklets) in the one-dimensionally recorded data. The proposed method, 2DAid, uses basic chromatographic laws to calculate the theoretical shape of a 2D peak (a cluster of peaklets originating from the same analyte) in order to define the area in which the peaklets of each individual compound can be expected to show up. Based on analyte-identity information obtained by means of mass spectral library searching, the individual peaklets are then combined into a single 2D peak. The method is applied, amongst others, to a complex mixture containing 362 analytes. It is demonstrated that the 2D peak shapes can be accurately predicted and that clustering and further processing can reduce the final peak list to a manageable size.     

Ultrasonic/microwave assisted extraction and diagnostic ion filtering strategy by liquid chromatography-quadrupole time-of-flight mass spectrometry for rapid characterization of flavonoids in Spatholobus suberectus

Spatholobus suberectus is a widely used herb in traditional medicine for the treatment of blood stasis syndrome and related diseases. In this work, a potential ultrasonic/microwave assisted extraction (UMAE) method was developed for efficient sample pretreatment, and a diagnostic ion filtering strategy with liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was established for rapid characterization of flavonoids in S. suberectus. The factors of UMAE influencing the extraction yield of flavonoids of S. suberectus were evaluated. The optimal conditions were determined as: microwave power of 300 W, extraction time of 450 s, 70% methanol as extraction solvent, solvent to solid ratio of 20 mL/g, ultrasound power of 50 W, extraction temperature of 80 °C, and one extraction cycle. Compared with commonly used extraction methods, UMAE showed higher efficiency and shorter extraction time for sample preparation. Subsequently, the major diagnostic ions and fragmentation pathways of flavonoids in Q-TOF-MS were summarized with available reference compounds. Using a new diagnostic ion filtering strategy, a rapid screening and identification of thirty-eight compounds was achieved in real S. suberectus samples. The results of this study clearly demonstrate the potential of UMAE for efficient extraction and LC-Q-TOF-MS for rapid and sensitive structural elucidation of flavonoids in S. suberectus, and open perspectives for similar studies on other medicinal herbs.     

Validation of a confirmatory method for the determination of melamine in egg by gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry

A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC-MS/MS, which was completed in 5 min for each injection. For the GC-MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC-MS and UPLC-MS/MS was 10 and 5 μg kg⁻¹, respectively. This validated method was successfully applied to the determination of melamine in real samples from market.