Dissimilarity analysis and automatic identification of monomethylalkanes from gas chromatography mass spectrometry data 1. Principle and protocols

Monomethylalkanes are common but important components in many naturally occurring and synthetic organic materials. Generally, this kind of compounds is routinely analyzed by gas chromatography mass spectrometry (GC–MS) and identified by the retention pattern or similarity matching to the reference mass spectral library. However, these identification approaches rely on the limited standard database or costly standard compounds. When unknown monomethylalkane is absent from the reference library, these approaches might be less useful. In this study, based on the fragmentation rules and empirical observation, many interesting mass spectral characteristics of monomethylalkanes were discovered and employed to infer the number of carbon atoms and methylated position. Combined with the retention pattern, a protocol was described for the identification of monomethylalkane analyzed by GC–MS. After tested by simulated data and GC–MS data of the gasoline sample, it was demonstrated that the developing approach could automatically and correctly identify monomethylalkanes in complicated GC–MS data.     

Identification of components in mixtures by a mathematical analysis of mass spectral data

Mathematical techniques for the identification of components in mixtures from the mass spectra of a series of related mixtures are described. The approach is analogous to library search methods in that spectra from a reference collection are compared with a multidimensional unknown. Searches are conducted with a library file containing approximately 17000 mass spectra. Results for the analyses of several mixtures are reported, to illustrate the effectiveness of the method.     

A computer system for structural identification of organic compounds from 13C NMR data

A computerised library search system for ¹³C NMR data is described. Given the spectral data of an unknown compound, the system will retrieve from the library those reference compounds exhibiting similar spectra. For comparison the spectral data are converted into a binary code, designed to reflect the underlying structure rather than exact values for chemical shifts. Thus, the ability of the system to retrieve compounds similar to the unknown (as opposed to identical) is greatly enhanced. A sophisticated search strategy adapting itself automatically to the problem at hand makes the system highly efficient.     

恩诺沙星粉中未知添加物的确证

应用非靶向分析技术,筛查、分析和确证恩诺沙星粉(水产用)中的非法添加物。分别制备甲酸酸化、碳酸钠碱化的恩诺沙星粉供试品溶液,经超高效液相色谱-二极管阵列检测器(UPLC-PDA)检测初筛,获取未知物色谱图。应用超高效液相色谱-飞行时间高分辨质谱(UPLC-TOF-HRMS),在正、负离子模式下对酸化、碱化样液进一步检测,获得未知物母离子和二级特征碎片的精确质荷比、同位素信息,并应用SCIEX OS软件进行分析推导。最后取疑似化合物对照品进行确证研究。UPLC-PDA初筛结果显示:酸化样液在1.870 min、5.122 min,碱化样液在5.122 min,均发现高响应未知色谱峰。对吸收波长和峰面积进行分析,推测含2个未知物,且未知物1(1.870 min)与未知物2(5.122 min)在酸性/碱性条件下可能发生转换。SCIEX OS软件分析推导结果显示:未知物2,母离子分子式拟合为C₁₁H₈O₂,二级碎片结构解析含1个苯环、2个羰基和1个通过成环连接的丙烯结构,推测为甲萘醌;未知物1的分子离子峰为C₁₁H₉O₅S^-,二级碎片仅采集到HSO₃^-,丢失部分与未知物2一致,结合甲萘醌常见衍生物类型,推测为亚硫酸氢钠甲萘醌。取甲萘醌、亚硫酸氢钠甲萘醌对照品进行对比研究,UPLC-PDA检测结果显示:未知物1与亚硫酸氢钠甲萘醌、未知物2与甲萘醌,保留时间和紫外光谱一致;向供试溶液中添加对照品溶液后检测,未知物为单一峰。UPLC-TOF-HRMS检测发现:未知物1与亚硫酸氢钠甲萘醌保留时间一致,一级质谱质量偏差为1.0×10^-6,二级谱库匹配度为100%;未知物2与甲萘醌保留时间一致,一级质谱质量偏差为0.6×10^-6,二级谱库匹配度为99.7%。未知物1和2的结构得以确证。亚硫酸氢钠甲萘醌可止血,与恩诺沙星治疗败血症的适应证相应,佐证试验结果。随着对兽药非法添加行为的严格监管和严厉打击,非法添加物和添加手段愈发隐蔽,常规靶向分析难以满足监控需求。该文详述的使用UPLC-PDA结合UPLC-TOF-HRMS对未知物进行非靶向分析的技术,可为药品、食品、保健品、化妆品及农药等产品中非法添加物的筛查和确证提供思路和技术参考。     

Chemical substructure identification by mass spectral library searching

A library-search procedure that identifies structural features of an unknown compound from its electron-ionization mass spectrum is described. Like other methods, this procedure first retrieves library compounds whose spectra are most similar to the spectrum of an unknown compound. It then deduces structural features of the unknown compound from the chemical structures of the retrievals. Unlike other methods, the significance of each retrieved spectrum is weighted according to its similarity to the spectrum of the unknown compound. Also, a “peaks-in-common” screening step serves to reduce search times and an optimized dot product function provides the match factor. If the molecular weight of the unknown compound is provided, the identification of certain substructures can be improved by including “neutral loss” peaks. Correlations between the presence of a substructure in a test compound and its presence among library retrievals were derived from the results of searching the NIST/EPA/NIH reference library with a 7891 compound test set. These correlations allow the estimation of probabilities of substructure occurrence and absence in an unknown compound from the results of a library search. This method may be viewed as an optimization of the “K-nearest neighbor” method of Isenhour and co-workers, with improvements that arise from spectrum screening, peak scaling, an optimal distance measure, a relative-distance weighting scheme, and a larger reference library.     

Perdeuterated n-alkanes for improved data processing in thermal desorption gas chromatography/mass spectrometry I. Retention indices for identification

The identification of organic compounds by GC/MS is useful in various areas such as fuel, indoor and outdoor air and flavour and fragrance applications. Multi-compound mixtures often contain isomeric compounds which have similar mass spectra and sometimes cannot be unambiguously identified by library search alone. Retention indices can help with confirmation of identification if they are reproducible. Using perdeuterated n-alkanes as a reference series for calculation of retention indices in GC/MS has a clear benefit because of the distinctive ion trace of m/z 34. Thermal desorption is useful for analysis of volatile organic compounds (VOCs) in air after sampling on appropriate sorbent cartridges. Comparison of indices between three systems, consisting of a thermal desorption unit, a gas chromatograph and a mass spectrometer, showed good agreement for compounds with well-defined peaks, whereas retention times varied.     

Fast gradient elution reversed-phase liquid chromatography with diode-array detection as a high-throughput screening method for drugs of abuse. II. Data analysis

In Part I of this work, we developed a method for the detection of drugs of abuse in biological samples based on fast gradient elution liquid-chromatography coupled with diode array spectroscopic detection (LC-DAD). In this part of the work, we apply the chemometric method of target factor analysis (TFA) to the chromatograms. This algorithm identifies the target compounds present in chromatograms based on a spectral library, resolves nearly co-eluting components, and differentiates between drugs with similar spectra. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguishes the present method from conventional library searching methods. Our library has a mean list length (MLL) of 1.255 and a discriminating power of 0.997 when both retention index and spectral factors are considered. The algorithm compares a library of 47 different compounds of toxicological relevance to unknown samples and identifies which compounds are present based on spectral and retention index matching. The application of a corrected retention index for identification rather than raw retention times compensates for long-term and column-to-column retention time shifts and allows for the use of a single library of spectral and retention data. Training data sets were used to establish the search and identification parameters of the method. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were found to be 92% and 94%, respectively.     

Characterization of impurities in sisomicin and netilmicin by liquid chromatography/mass spectrometry

The characterization of unknown impurities present in netilmicin and sisomicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. The volatile ion-pairing agent trifluoroacetic acid (TFA) was used for the retention of the main compounds and their impurities on a reversed-phase (RP) C18 column, because they are highly hydrophilic and basic compounds. The method showed good separation between netilmicin and its four potential related substances prescribed in the European Pharmacopoeia, which were identified by comparison of their retention times with those of the reference substances. Furthermore, in total 16 unknown impurities in a netilmicin sample and six in a sisomicin sample with unknown identity were detected. The structures of the unknown compounds were deduced based on comparison of fragmentation patterns with those of the reference substances investigated in LC/MSn experiments by the use of electrospray ion trap mass spectrometry.     

Computer-assisted multicomponent spectral analysis with fuzzy data sets

A new approach to the interpretation of spectra with “fuzzy sets” is described. A computer program CIF (Compound Identification with Fuzzy sets) is applied. This program is capable of finding components in a mixture by comparing the sample spectrum with reference spectra in a library. The applications discussed involve the interpretation of infrared spectra. The problems of spectral library search are discussed, an elementary introduction to fuzzy set theory is given, and applications to spectral library search are demonstrated.     

A new method for chemical identification based on orthogonal parallel liquid chromatography separation and accurate molecular weight confirmation

Recent advances in the theory and application of orthogonal LC separation have allowed for the establishment of a more effective method for the chemical identification of target compounds in complex samples, especially structurally similar compounds. In this study, a new chemical identification method based on orthogonal parallel separation and accurate molecular weight confirmation was developed. An orthogonal separation system consisting of an XTerra MS C(18) column, a home-made Click OEG column, and a Click CD column was established for separation and identification. In addition, 82 flavonoids were selected as references, to be used for the construction of a library. Retention times of each reference flavonoid on each column and accurate molecular weights were recorded and imported into a searchable library as "tags" for the unknown screening. For the method validation, two complex mixtures, fractions of Dalbergia odorifera T. Chen and Scutellaria baicalensis Georgi, specifically, were separated and identified. In total, nine compounds were unequivocally identified by retention time and confirmation of accurate molecular weight, demonstrating that this method is suitable and efficient for the chemical identification of complex samples.     

超高效液相色谱-四极杆飞行时间质谱法非靶向快速筛查进口粮谷中未知的农药残留

利用快速提取农药(fast pesticide extraction,FaPEx)方法前处理样品,并结合超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF)技术建立了非靶向快速筛查进口粮谷中未知的多种农药残留方法。采用1%(v/v)醋酸乙腈溶液提取粮谷中未知的农药残留,FaPEx固相萃取柱净化、浓缩,UPLC-Q-TOF检测。利用目标化合物特征离子的精确质量数、同位素匹配、二级碎片信息和保留时间进行数据库匹配,筛查可疑未知农药。结果表明,该方法无需对照标准品即可快速筛查进口粮谷中的农药残留,能够应用于进口粮谷样品的实际筛查。该方法快速、准确、分析通量高,可以为进口粮谷中农药残留的快速筛查和质量控制提供重要的方法依据。     

High-performance liquid chromatography systems for the separation of benzodiazepines and their metabolites

The high-performance liquid chromatographic (HPLC) retention characteristics of 21 benzodiazepine drugs and some of their metabolites have been examined on both silica and ODS-silica packing materials. Four HPLC systems have been considered and retention data are presented for the drugs on these systems. The correlation of retention data on the systems is considered with reference to the problem of identifying unknown benzodiazepines.     

Use of Retention Data as the First Step in the Identification of Cyclic Organic Peroxides in Temperature-Programmed Gas Chromatography

Temperature-programmed retention indices for eleven cyclic organic peroxides were determined by gas chromatography on slightly polar 5% biphenyl 95% dimethylpolysiloxane columns (DB-5 and Rtx-5MS) at three heating rates (5, 10, and 20° min⁻¹) from 60 to 300°C, using different chromatographs. Cyclic organic diperoxides and triperoxides had nearly constant retention indices when different heating rates and a short isothermal hold time (5 min) before the programmed increase in temperature were used. The usefulness of temperature-programmed retention indices was shown by using the data to predict the retention times and structures of unknown diperoxides or triperoxides derived from ketones. This is the first step in the identification of unknown cyclic organic peroxides, a process would otherwise require the availability of reference compounds. Revised: 7 and 17 November 2005     

Contribution to linearly programmed temperature gas chromatography. Further application of the Van den Dool-Kratz equation, and a new utilization of the Sadtler retention index library

Programmed temperature retention indices (PTRIs) calculated according to the equations of Van den Dool and Kratz, Golovnya and Uraletz, and Erdey et al. (also referred to as Antoine's integrated equation) are used in this work. Precalculation of isotherm retention indices from the results of a linearly programmed temperature GC is also presented. Deviations between experimental and calculated isothermal retention indices are below 2 retention index units. A relative "volatility" retention index is defined, as a function of the "volatilities"of the solute and the bracket reference n-alkanes. The comparison of the "volatility" retention indices with the PTRIs obtained with the other above equations shows absolute deviations of up to 4 retention index units. Based on an earlier "equivalent" temperature concept and on Tekler's proviso, a novel way for the utilization of Sadtler's retention index database, which takes advantage of the 3 data supplied by the library, is proposed.     

Analysis of gas chromatography—mass spectrometry data by reverse library search and the detection of substances not in the library

Computer least-squares methods are used to conduct an automatic reverse search of a library of complete mass spectra for the analysis of the output data matrix from a g.c.—m.s. system. The search is limited to those mass spectra falling within a narrow retention index window about the retention index corresponding to each scan in the datamatrix. The least-squares estimated parameters obtained are proportional to the concentrations of the substances present at each scan and are displayed graphically as the profiles of resolved overlapping g.c. peaks. The precision of the parameters is estimated. The root mean square Values of the residuals of the least-squares fits are used successfully to indicate the presence of substances not in the library. Examples are given of the results produced with data from this laboratory. Possible elaborations and improvements of this computer program are discussed.     

Similarity search for a 1H-NMR spectroscopic data base

A similarity-search system is described for proton-NMR spectroscopy. In order to achieve fast retrieval of reference compounds, the ¹H-NMR spectra of the data base and of the unknown are encoded in a bitsring. The individual bits of the binary signature describe different features of the spectra. Part of the coupling information is coded in such a way that effects of magnetic field strength are taken into account. The encoding thus permits a fast search for identical and structurally similar reference compounds in the data base even when the spectra were recorded at different magnetic field strengths. Because the search consists of weighted comparison of bits, each of them describing different spectral features, a choice of different kinds of searches is possible with the same signature by selecting appropriate weight vectors. Thus specific spectroscopic features can be selected for the search. Such a context-sensitive similarity-search system allows, for example, a search for compounds having similar multiplicities or similar subspectra in a given (e.g., aromatic) region of the spectrum. Furthermore, by adjusting two “software knobs” which influence the normalization of the search results, the user can choose between the two extremes of forward and reverse search, and between an identity search, similarity search or classification search. The results were tested on a small library containing 550 spectra including some mixtures and duplicates recorded under different experimental conditions at 250 and 400 MHz.     

Prediction of gas chromatographic retention indices of polychlorinated dibenzothiophenes on non-polar columns

Polychlorodibenzothiophenes (PCDTs) have been found in several kinds of environmental samples. The lack of reference compounds has meant that very little is known about their gas chromatographic behavior. Here we discuss the retention of 19 authentic PCDTs and their sulfones on the widely used gas chromatographic stationary phases DB-5 and DB-5ms. The retention order is different from that of the polychlorodibenzofurans. The data generated allowed us to carry out a multiple linear regression to generate parameters for predicting the retention indices of unknown congeners based only on their structural features.     

Retention time alignment of LC/MS data by a divide-and-conquer algorithm

Liquid chromatography-mass spectrometry (LC/MS) has become the method of choice for characterizing complex mixtures. These analyses often involve quantitative comparison of components in multiple samples. To achieve automated sample comparison, the components of interest must be detected and identified, and their retention times aligned and peak areas calculated. This article describes a simple pairwise iterative retention time alignment algorithm, based on the divide-and-conquer approach, for alignment of ion features detected in LC/MS experiments. In this iterative algorithm, ion features in the sample run are first aligned with features in the reference run by applying a single constant shift of retention time. The sample chromatogram is then divided into two shorter chromatograms, which are aligned to the reference chromatogram the same way. Each shorter chromatogram is further divided into even shorter chromatograms. This process continues until each chromatogram is sufficiently narrow so that ion features within it have a similar retention time shift. In six pairwise LC/MS alignment examples containing a total of 6507 confirmed true corresponding feature pairs with retention time shifts up to five peak widths, the algorithm successfully aligned these features with an error rate of 0.2%. The alignment algorithm is demonstrated to be fast, robust, fully automatic, and superior to other algorithms. After alignment and gap-filling of detected ion features, their abundances can be tabulated for direct comparison between samples.     

Extraction of information on the chemical structure of monofunctional compounds from retention data in gas—liquid chromatography by pattern recognition methods

A method for the determination of structural features of unknown compounds, based on multi-dimensional gas—liquid Chromatographic retention data, is presented. The species investigated are monofunctional compounds. The retention index is used as retention parameter. A two-step classification procedure is reported. The first step is the determination of a correction parameter for the retention index, the skeleton number. With these correction terms, the retention data are modified, and the second classification step for the determination of functional groups is executed with the modified data. The best classification obtained is based on the linear learning machine method. A 10-dimensional data set, i.e. the use of 10 stationary liquids, is sufficient for the total classification.     

Identification of unknown compounds on the basis of retention index data in comprehensive two-dimensional gas chromatography

The identification of unknown compounds in complex samples is very difficult. Comprehensive two-dimensional gas chromatography (GC x GC) provides very good resolution and improved identification reliability. Mass spectrometry is a powerful identification tool and retention index data are another good approach to this end. In this study, a second-order polynomial was used to calculate retention index data based on n-alkanes beyond the region of the 'isovolatile' curve in GC x GC, and the results in the 2nd dimension were validated by using the same stationary phase column in one-dimensional GC. To test the usefulness of the method, volatile compounds in a tobacco leaf extract fraction were analyzed using GC x GC, and 60 compounds were identified on the basis of their retention indices.