Liquid chromatography/ultraviolet/mass standards

This LC-MS experimental approaches used in NEF metabolite identification are also routinely employed for in vitro metabolism comparisons across species, in which liver microsomes or hepatocytes from humans and animal species are used. In addition, liver microsomal incubations followed by metabolite identification and quantitative estimation using liquid chromatography-ultraviolet/ mass spectrometry (LC-UV/MS) is an approach commonly taken to determine metabolic soft spots, where a major metabolic reaction takes place (Table 6.10). Use of UV detection allows for quantitative analysis of major metabolites in the absence of chemical standards, with the assumption that the metabolic reaction did not disturb the molecule s UV chromophore. 

The method for chloroacetanilide soil metabolites in water determines concentrations of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of alachlor, acetochlor, and metolachlor in surface water and groundwater samples by direct aqueous injection LC/MS/MS. After injection, compounds are separated by reversed-phase HPLC and introduced into the mass spectrometer with a TurboIonSpray atmospheric pressure ionization (API) interface. Using direct aqueous injection without prior SPE and/or concentration minimizes losses and greatly simplifies the analytical procedure. Standard addition experiments can be used to check for matrix effects. With multiple-reaction monitoring in the negative electrospray ionization mode, LC/MS/MS provides superior specificity and sensitivity compared with conventional liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/ultraviolet detection (LC/UV), and the need for a confirmatory method is eliminated. In summary,... 

BAA = 2-buto vacetic acid BE = 2-butoxyethanol BEA = 2-butoxyethanol acetate ECD = electron capture detector FID = flame ionization detection 6GC = gas chromatography HCl = hydrochloric acid HPLC = high performance liquid chromatography MS = mass spectrometry NaCI = Sodium chloride RSD = relative standard deviation UV = ultraviolet absorbance detection v/v = volume/volume w/v = weight/volume... 

CZE = capillary zone electrophoresis EC = electrochemical detector GC = gas chromatography HCD = Hall conductivity detector HPLC = high performance liquid chromatography IDMS = isotope dilution mass spectrometry MS = mass spectrometry RSD = relative standard deviation SEE = supercritical fluid extraction SPE = solid phase extraction UV = ultraviolet absorbance detection... 

As an approximation to a universal response detector, an ultraviolet (UV) detection with a high concentration of in vivo sample or in vitro incubation can be used to calibrate the mass spectrometric response of the metabolites. Josephs et al. (2009) recently reported the use of high-performance liquid chromatography (HPLC)-UV detection to get area responses of a 30-tiM in vitro microsome or hepatocyte incubation. The incubated sample was then diluted in matrix to create a single point calibration for mass spectrometric quantitation of the metabolites. The results from this method were successfully verified using buspirone and proprietary compounds for which the synthetic standards for their metabolites were available. 

We have included what we feel are the six currently most popular analytical techniques ultraviolet (UV) spectrophotometry, infrared (IR) spectrometry, proton nuclear magnetic resonance (NMR) spectrometry, mass spectrometry (MS), gas chromatography (GC), and high pressure liquid chromatography (HPLC). As we felt that the quality of data presented was of paramount importance in a reference source, we generated all of our data in our laboratory under uniform, reproducible conditions using state-of-the-art technology and verified chemical standards. 

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