Metabolites derivatization procedure

Detection in liquid chromatography is mostly performed by fluorescence and/or ultraviolet absorption. In a few instances, electrochemical detection has also been employed (357, 368). For compounds that exhibit inherent intense fluorescence such as albendazole and metabolites (319, 320, 338, 355), closantel (344), and thiabendazole and metabolites (378), fluorometric detection is the preferred detection mode since it allows higher sensitivity. Compounds that do not fluoresce such as eprinomectin, moxidectin, and ivermectin, are usually converted to fluorescent derivatives prior to their injection into the liquid chromatographic analytical column. The derivatization procedure commonly applied for this group of compounds includes reaction with trifluoroacetic anhydride in presence of A-methylimidazole as a base catalyst in acetonitrile (346, 347, 351, 352, 366, 369, 372-374). The formation of the fluorophore is achieved in 30 s at 25 C and results in a very stable derivative of ivermectin and moxidectin (353) but a relatively unstable derivative of eprinomectin (365). However, the derivatized extracts are not pure enough, so that their injection dramatically shortens the life of the liquid chromatographic column unless a silica solid-phase extraction cleanup is finally applied. 

After trying several different derivatization procedures, it was decided that for use as a monitoring tool it would be more practical to analyze for only one urinary component. The most logical choice was DBP. Both acaricides, DBA from CB, and DBH could all be converted to DBP. This could be extracted from the urine, and final detection of DBP could be accomplished by ECD/GC. No hydrolysis step per se was to be included in the procedure although it was felt certain that the hot H2SO4 would liberate any metabolites from the corresponding conjugates. 

One of the major advantages of LC-MS/MS over GC-MS or GC-MS/MS is that steroid hormones may be analyzed directly by LC-MS/MS without derivatization procedures, which are time-consuming and tedious [22,50-53], However, a number of studies demonstrated that the chemically derivatized steroid hormones were significantly more sensitive to LC-MS/MS detection than the underivatized hormones, because the neutral molecules of estrogens and metabolites might not be effectively ionized under electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes [4, 21, 25, 54, 55], In order to enhance the steroid hormone molecules sensitivity for LC-MS/MS analysis at pg/mL level, chemical derivatization is an effective technique for analysis of steroid hormones and metabolites. A list of derivatization reagents and application examples for steroid hormone analyses by LC-MS/MS and GC-MS are presented in Table 3. 

Some abuse drugs have been extracted from urine by SFE [viz. cocaine and its metabolites (134) and amphetamine and methamphetamine (135). In the first instance, the levels measured using SFE showed analyte recovery better than 70% for cocaine, better than 40% for benzoylecgonine, and better than 85% for ecgonine methyl ester from whole blood and urine. The limits of detection and quantitation were 1 and 10 ng, respectively, based on a 200-pL sample. Regarding amphetamine (AP) and methamphetamine (MA), an in situ SFE and chemical derivatization procedure followed by GC-isotope dilution mass spectrometry in urine was described. The mean recoveries achieved were 95% (RSD = 3.8%) for AP and 89% (RSD = 4%) for MA. The calibration graphs were linear within 100-500,000 ng/mL, varying the limits of detection and quantitation from 19 to 50 and from 21 to 100 ng/mL, respectively. 

The analysis of a large number of neurotransmitters and their metabolites as TMS denvatives has been excellently reviewed by Abramson and coworkers (1974). The electron impact (El) spectra of all the TMS-amino acids have also been reviewed (Iwase et al., 1979) In neurochemical studies, a number of workers have used the TMS derivatization procedure to measure tissue GABA (Cattabeni et al., 1976, 1977 Holdiness et al., 1981 Haseyawa et al, 1981). TMS-glutamate has been found to be extremely valuable for GC-MS analysis in the Cl mode with ammonia as the reagent gas (Dessort et al, 1982). An example of the use of TMS derivatives to monitor glutamate and aspartate release in brain dialysis experiments is presented in Fig. 4. 

Gas chromatography-mass spectrometry has proven to be a powerful analytical tool for detection of drugs of abuse in biological matrices. In particular, the use of the SIM mode has enhanced the sensitivity of the GC-MS method for detection of drugs of abuse. The versatility of the method has been extended by the use and development of novel derivatization procedures that have allowed a more diverse array of illicit drugs and their metabolites to be converted to thermally stable, volatile compounds. In addition, SPE and SPME methods also have improved the sensitivities of detection due to their capabilities for preconcentration of the targeted analytes. 

The same procedure, or modifications of it, was used by Zak et al (80), Talseth (42,82,82,83), and Haegele et al (46) for metabolic studies. Zak et al (80) point out that hydrolysis of conjugates of the drug may cause analytical results on biological samples to be variable, depending on the acid concentration during derivatization, and that selective analysis for unchanged hydralazine and acid-labile metabolites can be carried out by suitable adjustment of the acid concentration. 

Acidic pesticides and metabolites were concentrated from aqueous solution by the anion procedure of Richard and Fritz (10). The anionic materials in these concentrates were methylated using diazomethane and the derivatized products were separated and detected by gas chromatography. Test results of the recovery efficiencies by this method for several pesticides and suspected metabolites have been reported elsewhere (11). An overall recovery of 93% was achieved for sixteen acidic pesticides and metabolites spiked into water at 200 ppb. 

Only one gas chromatographic method has been reported to determine CCA in human plasma samples [53]. The standard metabolite used was the hydrochloride salt of CCA, while an analogous hydrochloride salt of the carboxylic acid was used as internal standard. Extraction by LLE method followed by SPE, and subsequent derivatization, was used to extract the metabolite of clopidogrel from human plasma and serum since this procedure could minimize matrix effects. In the LLE procedure, formic acid and diethyl ether were used to extract the analyte, followed by an SPE extraction of the residue in methanol using a Cis SPE column. 

Fourier transform ion-cyclotron (FT-ICR-MS) provides the highest mass resolution and accuracy, and enables the determination of the elemental compositions of metabolites, which facilitates annotation procedures for unknown compounds (95). Direct infusion analysis of plant extract without a previous separation and/or derivatization can be achieved however, its use is very restricted due to the equipment cost, the difficulties in hardware handling, and the extremely large amount of data generated. Takahashi et al. applied this technique to elucidate the effects of the overexpression of the YK1 gene in stress-tolerant GM rice (96). More than 850 metabolites could be determined, and the metabolomics fingerprint in callus, leaf, and panicle was significantly different from one another. 

For the purpose of achieving a selective SPE extraction that eliminates matrix components to the greatest extent possible without loss of analytes, an extensive optimization of the wash/elute conditions was conducted, the procedure is similar as described in previous examples. In order to achieve high efficiencies of extraction and reproducibility of quantification, a protein precipitation with ACN/methanol prior to SPE extraction is found important to assist releasing VitD metabolites from serum protein and derivatization by PTAD plays a critical role to improve the stability of VitD metabolites. 

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