9-Fluorenylmethyl chloroformate

Derivatization of primary and secondary amines using 9-fluorenylmethyl chloroformate to form a nonpolar, uv-absorbing derivative has been reported (90,91). Amphetamine and catecholamine were used as probes to evaluate this procedure. The derivatives were well behaved and allowed separation in a short time. 

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. 

Bartok, T., Szalai, G., Lorincz, Zs., Borcsok, G., and Sagi, F., High-speed RP-HPLC/FL analysis of amino acids after automated two-step derivatization with o-phthaldialdehyde/3-mercaptoproprionic acid and 9-fluorenylmethyl chloroformate, /. Liq. Chromatogr., 17, 4391, 1994. 

Fluorenyl carbanions, as initiators, 14 258 (+) -1 -(9-Fluorenyl)ethylchloroformate, chiral derivatizing reagent, 6 76t 9-Fluorenylmethyl chloroformate, molecular formula, 6 29It Fluorescamine, chemiluminescence reagent, 5 850... 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Amino acids derivatized with 9-fluorenylmethyl chloroformate (90) were separated by CE and determined by LIF with a pulsed laser LOD 0.5 nM (SNR 2)322. A sensitive technique for amino acids is capillary zone electrophoresis (CZE) combined with LIF of their fluorescein isothiocyanate (133) derivatives. Not all amino acids give good resolution. LOD for proline and arginine were 0.3 and 0.5 nM, respectively323. 

Bank RA, Jansen EJ, Beekman B and Te Koppele JM (1996) Amino acid analysis by reverse-phase high-performance liquid chromatography improved derivatization and detection conditions with 9-fluorenylmethyl chloroformate. Anal Biochem 240, 167-176. 

In liquid chromatographic analysis of macrolides and lincosamides, most popular is the ultraviolet detector (Table 29.4). Tylosin, tilmicosin, spiramycin, sedecamycin, and josamycin exhibit relatively strong ultraviolet absorption, but erythromycin, lincomycin, pirlimycin, and oleandomycin show extremely weak absorption in the ultraviolet region. Hence, detection at 200-210 nm has been reported for the determination of lincomycin (146). However, a combination of poor sensitivity and interference from coextractives necessitated extensive cleanup and concentration of the extract. Precolumn derivatization of pirlimycin with 9-fluorenylmethyl chloroformate has also been described to impart a chromophore for ultraviolet detection at 264 nm (140). 

C. Clapp, J. Swan, andj. Poechmann,/. Cbem. Educ. 69, A122-A126 (1992). Using 9-fluorenylmethyl chloroformate and HPLC to identify amino acids. 

The reagent is prepared by reaction of 9-fluorenylmethyl chloroformate (3, 145, 4, 237, 8, 230) with pentafluorophenol and triethylamine in ether (86% yield). 

PA Haynes, D Sheumack, LG Grieg, J Kibby, JW Redmond. Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate. J Chromatogr 588 107-114, 1991. 

H Anson-Moye, AJ Boning. A versatile fluorogenic labelling reagent for primary and secondary amines 9-fluorenylmethyl chloroformate. Anal Lett 12 25-35, 1979. 

J Kirschbaum, B Luckas, WD Beinert. Pre-column derivatization of biogenic amines and amino acids with 9-fluorenylmethyl chloroformate and heptylamine. J Chromatogr A 661 193-199, 1994. 

K Ou, MR Wilkins, JX Yan, AA Gooley, Y Fung, D Sheumack, KL Williams. Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate. J Chromatogr A 723 219-225, 1996. 

DT Blankenship, MA Krivanek, BL Ackerman, AD Cardin. High-sensitivity amino acid analysis by derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection applications in protein structure determination. Anal Biochem 178 227-232, 1989. 

B Carratu, C Boniglia, G Bellomonte. Optimization of the determination of amino acids in parenteral solutions by high-performance liquid chromatography with precolumn derivatization using 9-fluorenylmethyl chloroformate. J Chromatogr A 708 203-208, 1995. 

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. 

DN Heller. Determination of pirlimycin residue in milk by liquid chromatographic analysis of the 9-fluorenylmethyl chloroformate derivative. J AOAC Int 80 975-981, 1997. 

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). 

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