An integrated high-performance liquid chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases

Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 microL crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.     

气相色谱-质谱和液相色谱-质谱联用方法用于口腔癌代谢组学分析

口腔癌的发病率占全身恶性肿瘤的第6位,正确区分正常状态与良性和恶性口腔肿瘤,是恰当选择治疗方案的关键所在。本研究中,首先利用液相色谱-质谱和气相色谱-质谱联用方法分别得到健康人、良性口腔肿瘤患者和恶性口腔肿瘤患者血浆、尿液和唾液的代谢轮廓,然后应用正交信号校正的偏最小二乘法进行多变量统计分析。结果表明健康人、良性肿瘤患者和恶性肿瘤患者在血浆、尿液和唾液等3种体液代谢中都可以被区分开,而且找到和鉴定出19个重要差异代谢物。相关代谢通路分析显示,与健康人相比,良性和恶性口腔肿瘤患者都存在能量代谢紊乱和脂类代谢失衡的现象,但恶性口腔肿瘤患者还表现出三羧酸循环和肌醇代谢异常,这为临床诊断及治疗提供了重要信息。     

Characterization of N-acyl-D-biotinols by particle-beam liquid chromatography-mass spectrometry. An alternative to probe mass spectrometry for thermally labile samples.

In the course of preparing biotin-labeled nucleic acid probes, it was necessary to verify structures of intermediate N-acyl derivatives of biotinol. Characterization by mass spectrometry (MS) involved use of particle-beam liquid chromatography (LC)-mass spectrometry MS to supplement standard heated-solids probe techniques. The probe data for a sample of N-toluoylbiotinol indicated it to be a mixture of mono- and di-toluoylbiotinols which was inconsistent with other analytical information. Analysis of the same sample by LC-MS on a reversed-phase column with a water-acetonitrile gradient showed a single major peak with spectrum consistent with that for the monotoluoyl species. These results suggested that a thermal transacylation reaction might be occurring in the probe during heating prior to volatilization and ionization. This was confirmed by heating the sample to 200 degrees C and then repeating the LC-MS analysis to find peaks now present for biotinol and ditoluoylbiotinol as well as the starting material. These results demonstrate the value of particle-beam LC-MS as a technique for obtaining electron-impact mass spectra of thermally sensitive compounds.     

An entropy-based method for noise reduction of liquid chromatography-mass spectrometry data

Entropy-based methods have been extensively used to measure the uncertainty information in a variety of fields. In this article, a novel information theory-based method for reducing noise of liquid chromatography-mass spectrometry (LC/MS) data was developed. The uncertainty existed in the LC/MS chromatograms was captured and evaluated by information entropy. By comparing the information entropy computationally derived from mass chromatograms, the good quality chromatograms and the noisy chromatograms can be distinguished. The proposed method was applied in processing LC/MS data of “Jing-Zhi-Guan-Xin” troche which is a well-known preparation of traditional Chinese medicine (TCM). The obtained result indicated that this method is beneficial to reduce noise of LC/MS data of complicated chemical samples, such as TCM.     

High throughput log D determination using liquid chromatography-mass spectrometry

A method has been developed which allows direct measurement of partition coefficients (log D, log P) using liquid chromatography-mass spectrometry (LC-MS). The high throughput, microtiter plate based protocol uses small quantities of 10 mM analyte in DMSO solution (5 microL) and is therefore amenable to standard archive and screening formats. Single Ion Monitoring (SIM) mass spectrometry is used to achieve optimal sensitivity. Experimental log D values for 34 known drugs have been determined, with partition coefficients ranging from -2 to 5, giving data very similar to literature values. In these analyses, deviations from known values average less than 0.3 log units. The sample handling and data processing have been significantly automated, and the protocol has been applied to over 800 in-house lead molecules to date. In its format, sensitivity, throughput, and amenability to automation, it represents significant progress in the direct measurement of partitioning behavior [1].     

Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the online separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasaccharide differing from the acarbose in number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the offline 1- and 2-D NMR experiments, and atom assignment was made.     

Detection of Nepsilon-monomethyllysine using high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

Nepsilon-Monomethyllysine was identified in the serum, urine, brain, and liver samples of rats treated per os with L-deprenyl. The identification procedure included reaction with Fmoc chloride, clean-up, and analysis using HPLC-UV-MS. Oral administration of (-)-N-14C-methyl-N-propynyl(2-phenyl-1-methyl)ethylammonium hydrochloride L-deprenyl) to rats resulted in transfer of the radiolabelled methyl group to the Nepsilon-amino group of the endogenous lysine. The radiolabelled Nepsilon-monomethyllysine was urinary eliminated together with the other radiolabelled deprenyl metabolites, such as deprenyl-N-oxide and methamphetamine. The presence of Nepsilon-monomethyllysine has also been traced, and its concentrations were compared in the serum, liver and brain of rats subjected to L-deprenyl treatment. Methyl group transfer from the L-deprenyl to endogenous compounds; and the urinary elimination of their products may offer a vital way to eliminate or to decrease the degree of drug transmethylation to the lysine constituents of blood vessels' proteins.     

Purification of dinosterol for hydrogen isotopic analysis using high-performance liquid chromatography-mass spectrometry

A semi-preparative normal-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS) method is presented for the purification of various alcohol fractions from total lipid extracts derived from sediments, for the purpose of hydrogen isotopic measurement by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). 4-methylsterols, including the dinoflagellate-specific marker dinosterol (4,23,24-trimethylcholestan-22-en-3beta-ol), were successfully separated from notoriously co-eluting plant-derived pentacyclic triterpenoid alcohols and alkyl alcohols. We find that substantial hydrogen isotope fractionation occurs during chromatographic separation, demonstrating the importance of recovering the entire peak when subsequent hydrogen isotope analyses are to be performed. This is the first report of such hydrogen isotopic fractionation for a natural unlabelled compound.     

Structure elucidation of drug metabolites using thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry (LC-MS) has been used to provide structural information both from in vitro and in vivo experiments. This paper will describe the more salient aspects of the technique that have emerged. The ability of the interface to handle gradients was essential for its successful application to metabolism studies, owing to the wide range of compound polarity involved. The examples discussed in this paper include the use of LC-MS in the analysis of in vitro incubations of drugs with hepatocyte cell cultures and the direct analysis of plasma samples from in vivo studies in the dog.     

Separation of substituted fullerenes using non-aqueous reversed-phase liquid chromatography-mass spectrometry

A simple non-aqueous reversed-phase separation HPLC-MS method has been developed to allow for the rapid screening and separation of fullerenes and substituted fullerenes. This procedure provides confirmation that the proposed substitution methodology for the fullerene is not only successful but that multiple substitution products are obtained. The methodology is being expanded to analyze other substituted fullerene product mixtures.     

Enhanced monitoring of biopharmaceutical product purity using liquid chromatography-mass spectrometry

LC-MS is a widely used technique for impurity detection and identification. It is very informative and generates huge amounts of data. However, the relevant chemical information may not be directly accessible from the raw data map, particularly in reference to applications where unknown impurities are to be detected. This study demonstrates that multivariate statistical process control (MSPC) based on principal component analysis (PCA) in conjunction with multiple testing is very powerful for comprehensive monitoring and detection of an unknown and co-eluting impurity measured with liquid chromatography-mass spectrometry (LC-MS). It is demonstrated how a spiked impurity present at low concentrations (0.05% (w/w)) is detected and further how the contribution plot provides clear diagnostics of the unknown impurity. This tool makes a fully automatic monitoring of LC-MS data possible, where only relevant areas in the LC-MS data are highlighted for further interpretation.     

Development of generic liquid chromatography-mass spectrometry methods using experimental design

Standard approaches to development of liquid chromatography-mass spectrometry (LC-MS) methods, either ion-pairing or reversed-phase liquid chromatography, have been through trial and error or intentional variation of experimental factors. These approaches to method optimization fail to take into account interactions between experimental factors and therefore the results may not be optimal for the combination of experimental factors. Another approach to optimization is through the use of chemometrics. Chemometric approaches can be more efficient than trial and error or intentional variation because chemometrics make use of multivariate designs; experimental factors are varied simultaneously at the various levels. Therefore chemometrics can take into account interactions between factors. The goal of this study was to develop a generic ion-pair LC-MS method for the analysis of acidic compounds using a chemometric approach called design of experiments (DOE). Four acidic compounds which cover three classes of acidic functional groups: 1-naphthyl phosphate (1), 1-naphthalenesulfonic acid (2), 2-naphthalenesulfonic acid (3), and (1-naphthoxy)acetic acid (4) were used as model compounds to develop the generic method. This study illustrates that LC-MS conditions can be optimized efficiently with minimal amount of experimentation using a chemometric approach to experimental design.     

Quantification of amino acid ionic liquids using liquid chromatography-mass spectrometry

Amino acid ionic liquids (AAILs) containing imidazolium cations and amino acid (AA) anions, were synthesized and applied as task-specific ionic liquids. A sensitive and fast liquid chromatography-mass spectrometry (LC-MS) method was established for the quantitative analysis of 20 AAILs. Using ion pairing-reversed phase liquid chromatography technique, heptafluorobutyric acid was used as ion-pairing reagent to increase the retention of AAILs. Based on the zwitterionity of amino acid, this method was proposed to determine both the cation and the anion of AAILs simultaneously. The limit of detection of this method is down to 1-15ng/mL and the analysis time is less than 15min. According to the analytical data of seven selected AAILs, we found that the content of amino acid anion is always lower than that of butyl methyl imidazolium cation in AAILs. Moreover, the molar ratio of imidazolium cation to amino acid anion is dependent on the chemical property of the amino acid. These results supplied useful information on the interaction of imidazolium cation with acidic, basic, neutral and non-polar amino acids in AAILs.     

Ultrapure water for liquid chromatography-mass spectrometry studies

Improvements in trace enrichment techniques combined with the sensitivity of mass spectrometry offer enhanced opportunities to analyze ever lower concentrations of drugs, metabolites, pesticides or environmental pollutants. To perform HPLC and liquid chromatography-mass spectrometry (LC-MS) analyses under optimum conditions, the water used for mobile phase preparation needs to be highly purified and delivered on demand. Indeed, both UV photodiode array detection and MS detection methods are sensitive to organic contaminants (total organic carbon, TOC), and the water quality has a direct impact on the achievable detection limits. The benefits of UV photooxidation on TOC reduction for LC-MS studies were highlighted using electrospray ionization MS detection by comparing HPLC-grade bottled water, freshly produced UV185/254-treated water, and freshly produced non-UV-treated water.     

Matrix effects in pesticide multi-residue analysis by liquid chromatography-mass spectrometry

Three sample preparation methods: Luke method (AOAC 985.22), QuEChERS (quick, easy, cheap, effective, rugged and safe) and matrix solid-phase dispersion (MSPD) were applied to different fruits and vegetables for analysis of 14 pesticide residues by high-performance liquid chromatography with electrospray ionization-mass spectrometry (HPLC/ESI/MS). Matrix effect, recovery and process efficiency of the sample preparation methods applied to different fruits and vegetables were compared. The Luke method was found to produce least matrix effect. On an average the best recoveries were obtained with the QuEChERS method. MSPD gave unsatisfactory recoveries for some basic pesticide residues. Comparison of matrix effects for different apple varieties showed high variability for some residues. It was demonstrated that the amount of co-extracting compounds that cause ionization suppression of aldicarb depends on the apple variety as well as on the sample preparation method employed.     

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.     

Quantitative assessment of the reliability of identification by high-performance liquid chromatography-mass spectrometry

At present, mass spectrometry (MS) is the most reliable method for identification but there is not yet a quantitative equation describing this fact. In this investigation an approach to the quantitative assessment of the reliability of identification by MS is proposed which is useful for determination of the selectivity and the validation of analytical methods. Mass spectra of the analytes are presented as maps in which the characteristic ions and their intensities are used for identification. A formula for the quantitative expression of the significance of these parameters to the reliability and the identification is given. The contribution of the resolution of MS instruments or their possibilities of a multiple fragmentation to the reliability of the identification is shown. This approach makes it possible to compare the reliability of identification with different MS instruments. Despite the small contribution of the separation of the chromatographic column compared to the MS separation, the role of the column in the identification is very important to distinguish isomers because their MS spectra are similar.     

An objective comparison of pre-processing methods for enhancement of liquid chromatography-mass spectrometry data

Four data pre-processing methods have been applied with different settings to data sets obtained from the analysis of a pharmaceutical drug and its degradation products by liquid chromatography-mass spectrometry (LC-MS). The methods compared were the frequently used component detection algorithm (CODA) and three kinds of digital filters--matched filtration (MF), Gaussian second derivative (GSD) and Savitzky-Golay. The aim was to evaluate the performance and robustness of these methods for extracted ion chromatogram (XIC), total ion chromatogram (TIC) and base peak chromatogram (BPC) in the presence of different types of noise. In accordance with theory, the best improvements in signal-to-noise ratio (S/N) of the XICs were obtained with MF under the ideal case with random white noise. However, when highly coloured noise was present, it was found that no improvements in XIC S/N could be obtained with any of the pre-processing methods studied. GSD and CODA did, however, improve the S/N for both TIC and BPC. GSD and CODA also significantly reduced the background in the spectral domain, thereby facilitating the interpretation of the mass spectra. Another advantage associated with CODA and to some extent also with GSD is their data reduction ability.     

Radiochemical detection for packed capillary liquid chromatography-mass spectrometry

For the identification of trace level organic molecules, such as drug or pesticide metabolites, there is need of a practical method to do packed capillary liquid chromatography-mass spectrometry (LC-MS) with radiochemical detection. This problem has been successfully solved by use of a post column flow-splitter, with coaxially transported makeup flow that increases the split flow rate to a flow compatible with commercially available radiochemical flow cells. To test the device, 14C-labeled azoxystrobin, a commercial fungicide, was analyzed by liquid chromatography-radiochemical activity monitor-mass spectrometry (LC-RAM-MS) using a 0.32 mm i.d. packed capillary column. Azoxystrobin could be detected at 500 pCi with good signal/noise. The method is general and can be used with capillary LC columns of smaller diameters. Column efficiency of about 20,000 theoretical plates/m was achieved using either radiochemical or mass spectrometric data, thus demonstrating the lack of band broadening using the described method for radiochemical detection. The simple hardware described allows the routine use of packed capillary LC with radiochemical detection.     

Two-pump at-column-dilution configuration for preparative liquid chromatography-mass spectrometry

Preparative liquid chromatography-mass spectrometry (LC-MS) is widely used in parallel synthesis schemes to expedite purification. Recently, an alternative sample loading scheme, at column dilution, has been shown to dramatically increase the mass loading capacity of LC-MS purification methods. The prototype system utilized separate sample loading and binary gradient pumps. We report here a configuration for effecting at-column dilution using only the two pumps that provide the binary gradient flow. The advantages of a two-pump configuration are reduced cost, reduced space requirements, simplified control, and reduced service and maintenance issues. The two-pump at-column-dilution configuration is demonstrated for large- and small-scale LC-MS purifications. Purification on scales appropriate for high-throughput parallel synthesis can be achieved with small-scale chromatography using at-column dilution; purification of 20 mg of material is demonstrated using a 4.6 mm x 150 mm column and a flow rate of 3 mL/m. Reducing the scale of chromatography required for LC-MS purification has significant benefits, including the following. It requires less expensive columns, consumes less solvent, generates smaller-volume fractions (shorter dry-down time and the ability to collect into small-volume collector formats, such as 96-well plates), and has the potential for faster separations.