Fluorenylmethyl chloroformate, 9- fMOC

A derivatization with acid chlorides is also possible. Amino acids can be derivatized with 9-fluorenylmethyl chloroformate (FMOC) and separated on a CSP with X-cyclodextrin (ChiraDex gamma ), a cyclic oligosaccharide which consists of eight glucose units. 

A procedure to determine PIR residues in bovine milk using HPLC with the derivatization step for UV detection has also been published (211). The PIR was extracted from milk after protein precipitation and a two-step LLE procedure. The extract was evaporated to dryness, dissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (FMOC). The de-rivatized extract was analyzed by reversed-phase HPLC. Overall recovery was 89%, with 4% for coefficient of variation. A linear regression analysis of HPLC/UV results was compared with the HPLC/MS assay (209,210). The procedure takes about 2.5 hours to complete six or eight samples. Pirlimycin is stable in milk frozen to —60°C or below for at least 3 months. 

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). 

Leucine oxidized with "OH produces a 5 -hydroperoxy derivative which is subjected to chemical reduction to yield (2S)-y-hydroxyleucine, (2S,4S)-8-hydroxy-leucine, (2S,4R)-8-hydroxyleucine. The S -hydroxyleucines have been confirmed to be the reduction products of the corresponding hydroperoxyleucines. 5 -Hydro-xylysines are natural products formed by lysyl oxidase and therefore are not useful markers of radical-mediated oxidation. The other hydroxylysines are useful markers, however, with HPLC analysis of 9-fluorenylmethyl chloroformate (FMOC)... 

CAUTION 9-Fluorenylmethyl chloroformate (Fmoc-Cl, 17) can evolve CO2 (pressure ) upon prolonged storage therefore, bottles should be opened carefully. Fmoc-Cl is harmfiil if swallowed, inhaled, or absorbed through skin, is corrosive, and is a strong lachrymator. Inhalation may be fatal. 

A simple assay based on potent and specific inhibition of jack bean a-mannosidase has been devised for determining low concentrations of 162 (up to 0.5 cm ) in M anisopliae cultures (110). The new assay was used to demonstrate that the addition of L-lysine (163) to the culture medium stimulated production of the alkaloid by approximately fourfold. Other early metabolic precursors of 162 in this fungus, including a-aminoadipic acid, saccharopine (164), L-pipecolic acid (165), and L-lysine itself, were quantified by reverse-phase HPLC analysis of mycelial extracts derivatised with 9-fluorenylmethyl chloroformate (FMOC) (111). 

Pre-column derivatization—RPC analysis of phenylisothiocyanate (PITC), o-phthaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), or other derivatives of amino acids with UV or fluorescence detection is most common. This is the preferred methodology for life science research because of its higher sensitivity. More examples of precolumn derivatization of amino acids are shown in the life science section of this chapter. 

Both primary and secondary ammo acids react with 9-fluorenylmethyl chloroformate (FMOC chlonde) under alkaline, aqueous conditions (Anson-Moye and Boning, 1979). The reaction is complete in 30 s. However, although FMOC chloride is fluorescent, the excess reagent and the byproducts are easily removed by extraction prior to separation (Ejnarsson et al., 1983). 

The solubility of amino acids in CO2 modified with methanol, water and methylamine was increased sufficiently for SFC on columns packed with silica by deri-vatizing with 9-fluorenylmethyl chloroformate (FMOC) [16]. Valine, alanine, phenylalanine, lysine and stTine were separated (Figure 4) within 40 minutes. Derivatiza-tion with (-b)-l-(9-fluorenyl)ethyl chloroformate (FLEC) allowed separation of enantiomers more rapidly than by HPLC but with similar selectivity. 

Derivatization of sarcosine with 9-fluorenylmethyl chloroformate (FMOC) facilitates detection via fluorescence, but unfortunately the sarcosine derivatives are among the least stable of the amino acid derivatives formed this way, and the instability can lead to peak broadening. 

Table 3 Advances in the LC of precolumn derivatized AAs, obtained with phenylisothiocyanate (PITC), 5-dimethylaminonaphtalene-1-sulfonyl-CI (DANS), 4-dimethylaminoazobenzene-4-sulfonyl-CI (DABS), and with 9-fluorenylmethyl chloroformate (FMOC)... 

Figure 6 (details in Table 3) HPLC of AAs derivatized with 9-fluorenylmethyl chloroformate (FMOC). Peaks labeled with one letter abbreviations for protein AAs, as well as Hyp = hydroxyproline, R1 = FMOC-hydroxylamine, R2 = FMOC-hydroxide, R3 = reagent peak present in blank derivatization. (Reproduced with permission from Qu K et al. (1996) Journal of Chromatography A 723 219 Elsevier.)... 

Fluorenylmethyl chloroformate (FMOC) Primary and secondary amines 260 305... 

Related Reagents. 2-(ferf-Butylsulfonyl)-2-propenyl Chloroformate (Bspoc-Cl) l,l-Dioxobenzo[fi]thiophene-2-ylmethyl Chloroformate (Bsmoc-Cl) 9-Fluorenylmethyl Chloroformate (Fmoc-Cl) 2-(Methylsulfonyl)ethyl Chloroformate (Msc-Cl). ... 

Fluorenylmethyl chloroformate (FMOC) [17,18]) or special chromatographic conditions (e.g. ion-pair chromatography for determination of ionic compounds, like ciromazine and melamine, efhefon, or fosefyl [19-21]). 

Fluorenylmethyl chloroformate (FMOC) (Carpino et al., 1972) is another popular precolumn derivatization reagent. FMOC derivatization produces a very stable fluorescent product with both primary and secondary amines. The reaction is very straightforward. The sample is first mixed with a pH 7.7 borate buffer. Next the FMOC dissolved in acetone is added. The reaction is rapid, taking about 40 s. 

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