LC-MS method

USING CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) METHODS FOR THE DECISION OF MEDICAL PROBLEMS... 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

The issue of flow rate is of particular importance when a method is being developed to determine more than one analyte since the dependency of signal intensity on flow rate is likely to be different for each. This is demonstrated in the development of an LC-MS method for the analysis of a number of pesticides [3], the structures of which are shown in Figure 5.1. Initial experiments to determine the MS-MS transitions to monitor, shown in Table 5.2, and the optimum collision cell conditions were carried out by using flow-injection analysis. 

To allow all culture productiou to be coutrolled, a method for rapid analysis is required. Prior to development of an LC-MS method, the analysis was both complex and time-consuming, involving the purification of a relatively large amount of the antibody using affinity chromatography, enzymatic release, and subsequent derivatizafion of the oligosaccharides and their analysis by using capillary electrophoresis. 

More recently, liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) have been evaluated as possible alternative methods for carfentrazone-ethyl compounds in crop matrices. The LC/MS methods allow the chemical derivatization step for the acid metabolites to be avoided, reducing the analysis time. These new methods provide excellent sensitivity and method recovery for carfentrazone-ethyl. However, the final sample extracts, after being cleaned up extensively using three SPE cartridges, still exhibited ionization suppression due to the matrix background for the acid metabolites. Acceptable method recoveries (70-120%) of carfentrazone-ethyl metabolites have not yet been obtained. 

LC-MS is now a nature technology and operation of an LC-MS system is no longer the realm of an MS specialist. The proper choice of the LC-MS mode to be used in a specific situation depends on analyte class, sample type and problem (detection, confirmation, identification). On-line LC-MS is used more for specialised applications than for general polymer or rubber compound analysis. This derives from the fact that LC-MS method development (column, solvent system, solvent programme, ionisation mode) is rather time consuming. LC-MS (in particular with API interface) enables analysis of a wide range of polar and nonvolatile compounds which cannot be analysed by GC (icf. Scheme 7.7). 

A good starting point for LC-MS method development is a 50 x 2.1 mm Xterra MS Qg column (3.5 pm) injection volume 5 pL mobile phase H2O/ACN 90/10 to 10/90 v/v 250pLrnin 1 gradient 50 °C. Generic protocols and smart automated (or zero) method development is coming within reach [559]. 

There is no single LC-MS interface that is ideally suited for all compounds of interest to analytical chemists. It is evident that LC-APCI-MS and LC-PB-MS are currently the LC-MS methods most frequently used for polymer/additive analysis. The two techniques are compared in Table 7.69. When PB and API interfacing techniques are used, much more structural information can be obtained, and unambiguous identification... 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

The stoichiometry of the enzyme-inactivator complex has historically been most commonly determined using radiolabeled versions of the inactivator. Alternative methods include incorporation of a fluorescent or chromophoric group into the inactivator, or the use of quantitative LC/MS methods. 

The use of direct UV spectrophotometry to measure sample concentrations in pharmaceutical research is uncommon, presumably because of the prevalence and attractiveness of HPLC and LC/MS methods. Consequently, most researchers are unfamiliar with how useful direct UV can be. The UV method is much faster than the other methods, and this is very important in high-throughput screening. 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

Traditionally, the analysis of BFRs has been developed using GC as the principal separation technique, due to the volatility of these compounds. However, GC analysis of some BFR compounds, such as HBCD or TBBPA, presented some drawbacks. That because, in recent years, methods employing LC-MS and LC-MS-MS have been developed offering good results. Guerra et al. [112] presented an overview of current analytical methods for selected BFRs, focusing on instrumental determination using LC-MS. Table 1 summarizes different LC-MS methods found in the literature for the analysis of different BFRs. 

Suzuki et al. [480] developed a solid-phase extraction LC-MS method for determining pectenotoxin-2 and pectenotoxin-6 in seawater. These are toxins produced from toxic phytoplankton. 

The major limitation of high resolution accurate mass profiling is its inability to differentiate isomeric species with the same empirical formula. An example of isomers would be glucose (C6Hi206) and galactose (C6Hi206). In GC/MS and LC/MS methods, the isomers generally have different elution times that allow for... 

To identify a compound, five data points per peak may be sufficient. Quantitation may require at least 10 data points across a peak. Many of today s laboratories still house standard detectors (UV, ELSD, fluorescence, etc.) with maximum data acquisition rates at or below 20 Hz. Many conventional LC/MS methods acquire data at rates of 5 Hz or less. As shown in Figure 3.8, this is not sufficient for modem speed optimized chromatography. Obviously, selecting the wrong data acquisition rate will nullify all attempts to optimize chromatography. 

In the analysis of surfactants by MS in combination with interfacing techniques that were able to handle liquids, the FIA—MS and LC—MS methods performed by the API technique have become a powerful tool. No other analytical approach at that time was able to provide as much information as that obtainable with MS and MS-MS. The analysis of surfactants, even in complex mixtures on the one hand, is facilitated... 

However, large between-laboratory variabilities occurred that are indicative of poor reproducibility of methods. In so far as systematic trends could be observed, FIA-MS resulted more often in significantly lower concentrations than LC-MS or LC-FL methods. Within the LC-MS methods, the APCI interface yielded quantitative results which were invariably less than those obtained with LC-ESI-MS. This aspect is discussed in more detail in Chapter 4.3. LC-FL data were generally in fair-to-good agreement with LC-ESI-MS data. This held true for both LAS and NPEO analyses. 

It has been stated that these new RP-HPLC and APCI-LC-MS methods separate a higher number of pigments than previous ones. Although the pigment profiles may help the safe classification of the members of bacterium communities they alone are not suitable for the classification [292],... 

The Caco-2 permeability assay is usually performed in a Transwell device (Figure 18.1). The Transwell contains two compartments a donor and a receiver compartment. The apical donor compartment contains a porous membrane that supports the growth of the Caco-2 monolayer. Caco-2 cells are seeded on the porous membrane. Upon confluency of the cell culture, the compound is added into the donor compartment at a concentration range from one to several hundred micromolar. Samples are collected from the receiver compartment for up to 2 h, then LC-UV or LC-MS methods are used to quantify compound in each sample. The permeability coefficient of the compound is calculated based on the following equation ... 

Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. 

Hunt et al.150 proposed the incorporation of LC-MS techniques to eliminate other identity assays required in the QC lab. A LCQ ion trap mass spectrometer was added downstream of the UV spectrometer. In addition to a UV trace, a total ion current chromatogram was generated. Comparison to a reference chromatogram was performed to define identity without extensive interpretation of the mass spectral data. This LC-MS method eliminated the need for other identity assays such as N-terminal sequencing and was validated as a release assay for three proteins.150... 

Traditionally, HPLC, GC-MS, or LC-MS methods were used to monitor the clearance of small-molecule impurities. These analytical techniques often require unique solvents, columns, methods, reagents, detectors, and buffers for each analyte to be quantified. The NMR method, albeit not the most sensitive technique, normally does not have these problems. In this chapter, some examples will be used to demonstrate that NMR is a fast, generic, and reliable analytical technique for solving analytical problems encountered in the development of biopharmaceutical products. The NMR techniques described here require minimal sample handling and use simple standard NMR methods. They can easily be implemented and used for process development and validation purposes. 

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