9-Fluorenylmethyl chloroformate, derivatizing

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). 

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. 

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. 

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). 

Veuthey and Haerdi reported the separation of amphetamines using packed-column SFC [26]. The amphetamines were derivatized with 9-fluorenylmethyl chloroformate and chromatographed with a methanol or 2-propanol-modified carbon dioxide as the mobil phase. The separations were compared on bare silica and aminopropyl-bonded silica columns. Both columns gave comparable results and the separation of all five amphetamines (methylamphetamine, amphetamine, phenethylamine, ephed-rine, and norephedrine) was achieved in less than 5 min. Both methanol and 2-propanol-modified carbon dioxide gave comparable results. It was observed that the modifier concentration had more effect on the solvating power than the mobile-phase density. 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

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. 

Andres, J. V., Ealca, P. C., and Hernandez, R. H., Liquid chromatographic determination of aliphatic amines in water using solid support assisted derivatization with 9-fluorenylmethyl chloroformate, Chromatographia, 55, 129-135, 2002. 

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. 

Fluorenylmethyl chloroformate and its analogs (2-naphthyl chloroformate (NT-COCl), 9-fluorenyl-methyl chloroformate (FMOC-Cl), and 2-(l-pyre-nyl)ethyl chloroformate (PE-COCl)) are used as precolrunn derivatization reagents for HPLC. 

The essential shortage of an OPA/SH group reagent (reactive toward primary AAs only) can be eliminated by Shuster s principle - the automatic two-step precolumn derivatization method - by applying the OPA/MPA/fluorenylmethyl-chloroformate... 

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.)... 

Li, J. Milne, R.W. Nation, R.L. Tumidge, J.D. Coulthard, K. Johnson, D.W. A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 2001, 761, 167-175. 

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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