Derivatization, chemical reagents

Steroid hormones chemical derivatization, reagent selection and optimization Derivatization of steroids to enhance LC/MS detection, e.g., LSI1 /MS, A PCI VMS, and APPP VMS. [16, 17]... 

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Figure 27.18 Common configuration for postcolumn reactors with electrochemical analysis. (A) LC-chemical reaction-EC. Postcolumn addition of a chemical reagent (for example, Cu2+ or an enzyme). (B) LC-enzyme-LC. Electrochemical detection following postcolumn reaction with an immobilized enzyme or other catalyst (for example, dehydrogenase or choline esterase). (C) LC-EC-EC. Electrochemical generation of a derivatizing reagent. The response at the second electrode is proportional to analyte concentration (for example, production of Br2 for detection of thioethers). (D) LC-EC-EC. Electrochemical derivatization of an analyte. In this case a compound of a more favorable redox potential is produced and detected at the second electrode (for example, detection of reduced disulfides by the catalytic oxidation of Hg). (E) LC-hv-EC. Photochemical reaction of an analyte to produce a species that is electrochemically active (for example, detection of nitro compounds and phenylalanine). Various combinations of these five arrangements have also been used. [Reprinted with permission from Bioanalytical Systems, Inc.]...

Traditionally, most chemical methods for the direct determination of available lysine depended on the reaction of the e-amine with a chromophoric derivatizing reagent and then spec-trophotometric measurement. The most commonly employed chromophore is 1-fluoro-2,4-dinitrobenzene (FDNB), which was originally employed by Carpenter (102). Since then, numerous articles (103-106) have reported the direct (or indirect by difference) HPLC measurement of the FDNB-derivatized lysine. However, these methods can suffer from the problem that the reaction product of the FDNB-derivatized lysine (dinitrophenyllysine) is not entirely stable during acid hydrolysis (especially in the presence of carbohydrates) and correction factors are recommended (107). 

The detection of the individual C vitamers is complicated by their distinctly different properties. Although AA and DHAA are both ultraviolet (UV) absorbers, the absorbance maximum of DHAA is between 210 and 230 nm (15,18,42,43). For practical detection purposes, this makes DHAA particularly susceptible to interferences from a number of naturally occurring food constituents and limits the choice of reagents and solvents. In contrast, AA exhibits a pH-dependent absorbance maximum of 245-265 nm, which makes UV absorbance an ideal choice for detection. On the strength of its reducing capacity, AA can be detected electrochemically, but DHAA is electrochemically inactive. Neither AA nor DHAA fluoresce naturally. However, DHAA readily forms a fluorescent quinoxaline derivative upon reaction with o-phenylenediamine. As a result, chemical derivatization is often used to achieve the sensitivity needed to detect the naturally occurring vitamin C in food. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

In-situ chemical derivatizations were performed by adding 0.5 to 2 mL of the derivatization reagent, trimethylphenyl ammonium hydroxide (TMPA) in methanol (Eastman Kodak Company, Rochester, NY) directly to the sample cell of an ISCO model SFX extraction unit. The sample and reagent were pressurized with C02 (typically 400 to 500 atm), and heated to an appropriate temperature (typically 80°C). The derivatization was performed in the static SFE mode for 5 to 15 minutes, then the derivatized analytes were recovered by dynamic SFE using a typical flow rate of 0.6 to 1.0 mL/min (measured as liquid C02 at the pump). No other sample preparation of the derivatized extracts was performed prior to GC analysis. All GC analyses were performed using Hewlett-Packard 5890 GCs with appropriate detectors and HP-5 columns (25 m X 250 pm i.d., 0.17 pm film thickness). 

In precolumn derivatization, the derivatization process alters the chemical nature of the analytes. Therefore, it may be necessary to develop new chromatographic methods. However, the separation can be optimized for the particular analytes, and any excess reagent can be removed so that it does not interfere with detection. The selection of precolumn derivatization reagents is therefore less restricted than is the choice of postcolumn derivatization reagents, and rapid kinetics are not particularly important. The stability of the derivative is important, however, as is the percent derivatization, which should be as near to 100% as possible. It is also important that the reaction yield only one derivative per analyte, so that coelutions of extra peaks does not occur, and so solute identification and quantitation are accurate. 

Chemical derivatization of the capillary wall has also been used to alter the properties of the interface. Derivatization using trichlorovinylsilane followed by copolymerization with polyacrylamide is one approach. Another effective reagent is (o -methacryloxypropyl)trimethoxysilane, followed by cross-linking the surface-bound methacryl groups with polyacrylamide. 

Chemical derivatization is a standard procedure for GC-MS analysis of steroid hormones, because steroid hormones are not volatile to go through GC column [1, 6], The major concerns of derivatization reagents for GC-MS analysis of steroid hormones are the completeness of the derivatization reaction and the volatility of hormone derivatives. The typical derivatization reagents for GC-MS samples are silylation reagents, e.g., Af-methyl-Af-trifluorotrimethyl acetamide (MSTFA, [37, 39]) and A,0-bis(trimethylsilyl) trifluoroacetamide (BSTFA, [33]), which react with both alcoholic and phenolic hydroxy groups on steroid hormone molecules. 

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