ANALYTICAL BIOCHEMISTRY 240, 167–176 (1996)
ARTICLE NO. 0346
Amino Acid Analysis by Reverse-Phase High-Performance
Liquid Chromatography: Improved Derivatization and
Detection Conditions with 9-Fluorenylmethyl Chloroformate
Ruud A. Bank, Evelien J. Jansen, Bob Beekman, and Johan M. te Koppele1
TNO Prevention and Health, Division of Vascular and Connective Tissue Research,
P.O. Box 2251, 2301 CE Leiden, The Netherlands
Received October 23, 1995
An improved method for the quantitative derivatiza-
tion of amino acids with fluorenylmethyl chloro-
formate (FMOC-Cl) is described. Amino acids are de-
rivatized in borate buffer at pH 11.4 for 40 min at
ambient temperature. All amino acids resulted in sta-
ble derivatives. In particular, improved derivatization
was obtained with the troublesome amino acids His
and Tyr: exclusively monosubstituted His and disub-
stituted Tyr were formed, eluting as free peaks in the
chromatogram. These derivatives show a higher fluo-
rescence response than their disubstituted and mono-
substituted counterparts, respectively, resulting from
other protocols. Under the new conditions, consider-
able less of the hydrolysis product of FMOC-Cl is seen
in the chromatograms. Baseline noise was substan-
tially reduced at a higher emission wavelength (630
nm instead of 313 or 340 nm). With simple precautions,
extensive adsorption of the disubstituted derivatives
(Lys, Hyl, and Tyr) on plastic or glass surfaces could
be prevented. Calibration curves were linear over a 10
to 300 molar ratio of FMOC-Cl to total amino acid. The
detection limits are in the femtomole range and the
derivatives are stable for more than 48 h, thus permit-
ting automated analysis of multiple samples. q 1996
Academic Press, Inc.
Although numerous precolumn derivatization re-
agents have been introduced for the analysis of amino
acids (1–4), all of them show various shortcomings. The
ideal reagent should react rapidly and quantitatively,
under mild conditions, with both primary and second-
ary amino acids, without being disturbed by sample
matrix components such as salts. Furthermore, the re-
1
To whom correspondence should be addressed. Fax: 31-71-
5181904.
0003-2697/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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agent should give rise to a single, stable derivative per
amino acid. The derivative should be detectable with
high sensitivity and the reagent itself or its degrada-
tion products should not interfere with the chromato-
graphic separation. 9-Fluorenylmethyl chloroformate
(FMOC-Cl)2 meets most of these criteria. This label
was introduced for amino acid analysis by Einarsson
et al. (5, 6). FMOC-Cl reacts rapidly and quantitatively
with both primary and secondary amino acids under
mild conditions and is relatively insensitive for salts.
The resulting carbamates exhibit high fluorescent re-
sponse (detection at the low picomole level), are stable
at room temperature for several days, and show excel-
lent chromatographic behavior on reversed-phase co-
lumns.
A disadvantage of FMOC-Cl is its reactivity toward
water; after hydrolysis and decarboxylation, the fluo-
rescent alcohol, FMOC-OH, elutes in the middle of the
chromatogram. At high concentrations, FMOC-OH
overlaps with other amino acids in the chromatogram,
complicating the quantification of these amino acids.
Inasmuch as FMOC-Cl is also fluorescent, excess re-
agent should be removed before chromatography, for
instance by extraction with organic solvents. With such
extractions part of the FMOC-OH is also removed (5–
15). To avoid the cumbersome extraction step, excess
FMOC-Cl can be eliminated with 1-adamantylamine
(8, 9, 15–18) or hydroxylamine (19, 20). A disadvantage
is that the amount of FMOC-Cl added and reaction
2
Abbreviations used: FMOC-Cl, 9-fluorenylmethyl chloroformate;
FMOC-OH, hydrolysis product of FMOC-Cl with water; Hyl, hydrox-
ylysine; Hyp, hydroxyproline; N-FMOC-His, monosubstituted deriv-
ative of histidine (a-amino); N-FMOC-Tyr, monosubstituted deriva-
tive of tyrosine (a-amino); N,NH-FMOC-His, disubstituted deri-
vative of histidine (a-amino and imidazole NH); N,O-FMOC-Tyr, di-
substituted derivative of tyrosine (a-amino and phenolic hydroxyl
group).
167
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168 BANK ET AL.
time should be controlled carefully, since FMOC-OH
remains in the reaction mixture.
Other specific drawbacks of FMOC-Cl are the low
fluorescent properties of the Cys derivative as well as
that of the di-FMOC derivative of His and the mono-
FMOC derivative of Tyr (11, 19, 20). Furthermore,
under the routinely used reaction conditions, Tyr is
converted into a mono- and a disubstituted form (N-
FMOC-Tyr and N,O-FMOC-Tyr, respectively). Clearly,
the presence of two peaks from one amino acid compli-
cates its quantification. The disubstituted form of His
(N,NH-FMOC-His) is not stable at room temperature
and is gradually converted to the monoderivative (N-
FMOC-His), again complicating quantification (5, 11).
We have studied the derivatization conditions in
great detail and present a reproducible protocol for
amino acid analysis. In particular, His is present only
as a monoderivative, whereas Tyr is converted exclu-
sively into N,O-FMOC-Tyr. These derivatives show
higher fluorescence properties than their di- and mono-
substituted counterparts, respectively, allowing more
unambiguous and more sensitive detection. Interest-
ingly, the new protocol results in smaller amounts of
the fluorescent hydrolysis product of FMOC-Cl with
water, FMOC-OH, thus minimizing the interference of
this compound with other amino acids in the chromato-
gram. Finally, monitoring of emitted light at 630 in-
stead of 313 or 340 nm resulted in a decreased baseline
noise, thus improving the detection limit.
MATERIALS AND METHODS
Reagents; preparation of amino acid standard. Ace-
tone, tetramethylammonium chloride, boric acid, and
sodium hydroxide were purchased from Merck (Darm-
stadt, Germany). HPLC-grade acetonitrile was ob-
tained from Rathburn (Walkerburn, Scotland). Sodium
azide (Baker grade) and citric acid were from Baker
(Deventer, Holland); 9-fluorenylmethyl chloroformate
was supplied by Fluka (Buchs, Switzerland). Unless
stated otherwise, all reagents were of analytical grade.
For derivatizations, the amino acid standard for colla-
gen hydrolysates (A-9531) from Sigma (St. Louis, MO)
was used. This standard contains 2.5 mmol of each
amino acid per milliliter, except for L-cystine (1.25
mmol per milliliter) and L-proline and hydroxy-L-pro-
line (12.5 mmol per milliliter). Hydrolysis was per-
formed on human angiotensin-II and bovine serum al-
bumin (Sigma) as well as on lysozyme (Boehringer,
Mannheim, Germany). Bovine type II collagen was
purified from nasal cartilage according to Miller
et al. (11).
Borate buffer was prepared from boric acid (0.1 M)
adjusted to the desired pH with 5 N sodium hydroxide.
The routinely used amino acid standard was prepared
by adding 120 ml of the stock solution to 100 ml 0.1 M
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borate buffer (pH 8.0 or 11.4) containing 6 mM homoar-
ginine (internal standard).
Derivatization. For derivatization, the reactant so-
lution consisted of FMOC-Cl dissolved in acetone (1.5
mg/ml, 6 mM). Two hundred microliters of FMOC solu-
tion was added to 200 ml of an amino acid standard
solution prepared as described above, mixed immedi-
ately, and then allowed to stand at room temperature
for 10 min (pH 8.0) or 40 min (pH 11.4). Termination
of the reaction and removal of excess reagent (FMOC-
Cl), its hydrolysis product FMOC-OH, and acetone
were performed by extraction with 600 ml pentane.
After mixing and a short period (around 0.5 min) to
allow phase separation, the upper layer was discarded.
After two additional extractions, 400 ml 25% (v/v) aceto-
nitrile in 0.1 M borate buffer, pH 8.0 (for the derivatiza-
tion reaction performed at pH 8.0), or 400 ml 25% (v/v)
acetonitrile in 0.25 M boric acid (pH approximately 5.5)
(for the derivatization reaction at pH 11.4) was added.
A 50-ml aliquot of the diluted sample was injected into
the HPLC system; thus, 50 pmol of most of the amino
acids (exceptions: Hyp and Pro 250 pmol, homoarginine
100 pmol, Cys 25 pmol) was applied on the column. In
this procedure a 75-fold excess of the fluorescent label
is present.
Instruments. The HPLC system (Separations, Hen-
drik Ido Ambacht, The Netherlands) consisted of a
Gynkotek Model 480 multisolvent delivery system, a
Sparks Holland Triathlon autosampler, a Jasco Model
821-FP fluorometer, and a Lab-Quatec Model Gastorr
GT-103 degasser. Peak Master (Harley Systems) soft-
ware, run under Microsoft Windows version 3.11, was
used for data acquisition and processing. The reversed-
phase HPLC column (4.6 1 150 mm; Micropak ODS-
80TM; 5-mm spherical silica particles with 80 Å pore
size; Varian) was thermostated at 407C with a HPLC
column water jacket (Alltech, Deerfield, IL).
Chromatography. Derivatized amino acids were
separated using a slightly modified ternary gradient
system described by Cunico et al. (10) and Miller et al.
(11). Solvent A was 20 mM citric acid containing 5 mM
tetramethylammonium chloride and 0.01% (w/v) so-
dium azide, adjusted to pH 2.85 with 20 mM sodium
acetate containing 5 mM tetramethylammonium chlo-
ride and 0.01% (w/v) sodium azide. Solvent B was 80%
(v/v) of 20 mM sodium acetate solution containing 5
mM tetramethylammonium chloride and 0.01% (w/v)
sodium azide (adjusted to pH 4.5 with concentrated
phosphoric acid) plus 20% (v/v) methanol. Solvent C
was acetonitrile. For elution the gradient was used as
given in Table 1. The flow rate was maintained at 1.4
ml/min throughout the analysis. The separation was
performed at a column temperature of 407C and fluo-
rescence was monitored at 254 and 630 nm (excitation
and emission wavelengths, respectively).
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 9-FLUORENYLMETHYL CHLOROFORMATE AMINO ACID ANALYSIS 169

TABLE 1 Derivatization reaction time. At pH 8.0, the reac-

Chromatographic Gradient Conditions for HPLC Analysis tion of FMOC-Cl with amino acids and water proceeds
of FMOC Derivatized Amino Acids rapidly: essentially no increase in the amount of deriva-
tized amino acids or FMOC-OH is seen after 2 min,
Time Eluent A Eluent B Eluent C including N,NH-FMOC-His (Fig. 2). The acidic amino
(min) (%) (%) (%)
acids Asp (data not shown) and Glu are the slowest
0 75 — 25 reacting amino acids, resulting in a significantly lower
11.5 60 — 40 derivative yield at the early time points. A slow reac-
13 60 — 40 tion is also seen with the imidazole group of His and,
13.1 — 64 36
18 — 62 38
especially, the phenolic hydroxyl group of Tyr. In the
25 — 30 70 beginning (0.5–4 min) the monosubstituted form of Tyr
30 — 25 75 predominates. At around 10 min, less N-FMOC-Tyr is
32 — 25 75 present and N,O-FMOC-Tyr is the major species.
32.1 75 — 25 Amounts of disubstituted Tyr still increase for a long
40 75 — 25
time, concomitant with a decrease in amounts of mono-
substituted Tyr. Therefore, at pH 8.0 the time point
at which the reaction is stopped should be carefully
RESULTS
controlled. Because of formation of unknown com-
Formation of His and Tyr derivatives and FMOC- pounds interfering with the late-eluting amino acids in
OH. Figures 1A–1D show chromatograms of four dif- the chromatogram, the reaction cannot proceed for a
ferent FMOC derivatizations. Chromatograms A and longer time (Fig. 1B).
B are derivatized for 2 and 40 min in 0.1 M borate At pH 11.4 the reaction of FMOC-Cl with amino acids
buffer, pH 8.0. Chromatograms C and D are compara- and water proceeds even more quickly, as can be seen
ble with A and B, respectively, the exception being that from the peak area of Glu and N,O-FMOC-Tyr (Fig. 2).
the pH of the borate buffer was 11.4. As for pH 8.0, no increase in the amount of derivatized
Apart from an increase of unknown peaks eluting amino acids is seen after 2 min. The only exception is
between 23 and 35 min with prolonged derivatization N-FMOC-His: with time its amount increases. At the
time, no differences are seen between chromatograms same time, the amount of N,NH-FMOC-His decreases.
A and B. At pH 8.0 His is present as the disubstituted The decrease of N,NH-FMOC-His is due to the selective
derivative and elutes between Hyl and Lys in a region release of the FMOC label from the imidazole group,
of somewhat increased baseline noise. Barely any or caused by the high pH. After 40 min barely any N,NH-
no N-FMOC-His (the first peak in the chromatogram) FMOC-His is detectable and subsequently no increase
is detected. Tyrosine is present both as N,O-FMOC-Tyr is observed in the amount of the monosubstituted form.
and as N-FMOC-Tyr. The monosubstituted form of Tyr Since all amino acids are quantitatively derivatized
elutes after Ala, whereas N,O-FMOC-Tyr elutes at the at pH 11.4 after a short period (2 min), the reaction
very end of the chromatogram. could in theory be stopped at this point. However, at
A completely different situation is seen with respect this time point His is present as two derivatives; fur-
to the amount of FMOC-OH (the hydrolysis product of thermore, a large FMOC-OH peak is seen in the chro-
FMOC-Cl with water) and the formation of His and matogram (Fig. 1C). Increasing the derivatization time
Tyr derivatives for the two time points at pH 11.4 (chro- to 40 min resulted in a quantitative conversion of
matograms C and D). After a reaction period of 40 min, N,NH-FMOC-His into N-FMOC-His (Figs. 1D and 2)
less FMOC-OH is seen compared to the 2-min period.
and, surprisingly, in a decrease of the amount of
After 2 min, Tyr is essentially present as N,O-FMOC-
FMOC-OH (Fig. 1D). The decrease in the amount of
Tyr, whereas His is seen as both the mono- and disub-
FMOC-OH, which is in the order of 30%, results in a
stituted derivative. After 40 min, His is almost quanti-
more accurate quantification of Ala (compare Figs. 1C
tatively converted to N-FMOC-His, which elutes in a
and 1D), whereas the resolution of Gly with FMOC-
clean region at the very beginning of the chromato-
OH remains unaffected. The reaction must be stopped
gram. Interestingly, Tyr is still present as N,O-FMOC-
at around 40 min; reaction times longer than 60 min
Tyr. The disubstituted form of Tyr shows a higher
result in a significant conversion of N,O-FMOC-Tyr
fluorescence response than its monosubstituted coun-
into N-FMOC-Tyr and in the hydrolysis of the FMOC
terpart. For His the opposite is seen: N-FMOC-His
label from other amino acids as well.
shows a higher fluorescence signal than N,NH-FMOC-
His. No increase of unknown peaks between 23 and 35 Effect of amino acid concentration. Linearity of the
min was observed with prolonged derivatization time. relationship between peak area and amino acid concen-
These findings initiated a more in-depth study of the tration was determined with standard amino acid mix-
derivatization procedure. tures in 0.1 M borate, pH 8.0 or 11.4, in concentrations

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170 BANK ET AL.

FIG. 1. Elution profiles of amino acid standards derivatized by reaction with FMOC-Cl. Each amino acid represents 50 pmol, except Hyp
and Pro (250 pmol), homoarginine (100 pmol), and Cys (25 pmol). (A and B) Derivatization reaction performed at pH 8.0 for 2 and 40 min,
respectively; (C and D) derivatization reaction performed at pH 11.4 for 2 and 40 min, respectively. The peaks are labeled by one-letter
codes for the usual protein amino acids; IS, internal standard (homoarginine); OK, hydroxylysine; OP, hydroxyproline; H7 and Y7, monosubsti-
tuted forms of His and Tyr, respectively.

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 9-FLUORENYLMETHYL CHLOROFORMATE AMINO ACID ANALYSIS 171

FIG. 2. Peak area versus reaction time at room temperature for the derivatization of amino acids with FMOC-Cl at pH 8.0 and pH 11.4.
For purposes of clarity only representative amino acids are shown.

ranging from 113 to 3600 mM (resulting in a molar
ratio of FMOC-Cl to total amino acid from 9.6 to 300).
Linear calibration curves were obtained for all amino
acids at pH 11.4 (Fig. 3) (r ú 0.999). The same situation
is seen for pH 8.0, the only exception being Tyr (r Å
0.995) (Fig. 3). A linear response is seen for N,O-
FMOC-Tyr and N-FMOC-Tyr at pH 8.0 at a molar
FMOC-Cl/amino acid ratio between 18.8 and 300. Be-
low a ratio of 18.8, a significant decrease of the disubsti-
tuted form and an increase of the monosubstituted
form of Tyr are routinely seen at pH 8.0. This is most
likely caused by the slow reaction of the fluorescent
label with the phenolic hydroxyl group of Tyr in com-
parison to the rapid consumption of FMOC-Cl by the
other, faster-reacting amino acids.
Reproducibility of derivatization and chromatogra-
phy. The reproducibility of derivatization at pH 11.4
was established by analyzing 10 different derivatiz-

FIG. 3. Linearity between different concentrations of amino acids and fluorescence of the corresponding derivatives after derivatization
with FMOC-Cl at pH 8.0 and 11.4 for 40 min at room temperature; only representative amino acids are shown.

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172 BANK ET AL.

TABLE 2
Intraday Precision on Repeated Determinations of Individual Amino Acids on a Standard Mixture

Intraassay variabilitya Interassay variabilityb

RT %SD Area %SD RMR %SD RT %SD Area %SD RMR %SD

His 7.07 0.13 127 1.34 1.23 0.87 7.07 0.18 123 2.15 1.23 1.69
Arg 8.58 0.10 104 1.31 1.01 0.83 8.58 0.15 101 2.04 1.01 0.89
ISc 10.02 0.14 103 1.41 — — 10.02 0.13 100 1.97 — —
Hypd 11.07 0.12 122 1.35 1.18 0.59 11.07 0.09 119 2.39 1.19 2.15
Ser 11.37 0.09 120 1.13 1.17 0.86 11.38 0.08 117 3.58 1.17 3.49
Asp 12.05 0.10 104 1.68 1.01 1.07 12.06 0.08 101 1.91 1.01 1.99
Glu 12.42 0.09 106 1.51 1.02 0.83 12.43 0.07 103 1.50 1.03 2.30
Thr 13.27 0.08 108 1.33 1.05 0.80 13.27 0.06 105 1.13 1.05 1.72
Gly 14.11 0.08 130 4.40 1.26 4.93 14.12 006 125 3.83 1.25 3.78
Ala 15.59 0.03 128 0.97 1.24 1.03 16.00 0.03 124 2.29 1.24 2.56
Pro 17.18 0.05 129 1.08 1.25 1.08 17.19 0.03 126 1.75 1.26 2.19
Met 18.29 0.04 91 1.47 0.88 0.74 18.31 0.09 89 1.56 0.89 2.22
Val 19.16 0.05 126 1.56 1.21 1.17 19.18 0.11 121 1.57 1.21 2.09
Phe 20.58 0.06 124 1.20 1.20 0.80 21.02 0.13 122 1.77 1.22 2.32
Ile 21.39 0.07 116 2.39 1.12 1.46 21.41 0.04 114 1.59 1.14 2.43
Leu 21.53 0.06 133 1.71 1.29 1.81 21.56 0.09 132 1.98 1.32 2.08
Cys 23.05 0.06 49 2.34 0.24 2.13 23.07 0.09 49 2.71 0.49 2.70
Hyle 24.07 0.07 202 1.10 1.95 0.99 24.08 0.08 195 1.11 1.95 2.06
Lys 25.40 0.06 228 0.68 2.21 1.42 25.42 0.07 225 1.00 2.25 2.11
Tyr 26.49 0.05 167 2.80 1.62 4.04 26.50 0.07 173 1.86 1.73 3.29
Mean 0.08 1.64 1.44 0.09 1.98 2.32

Note. RT, retention time (in minutes / seconds; 8.58 Å 8 min and 58 s); values are the mean (n Å 10). RMR, relative molar response
(values relative to homoarginine Å 1); values are the mean (n Å 10). SD, standard deviation. Area, area/pmol amino acid (expressed in
arbitrary units); values are the mean (n Å 10).
a
Precision of the chromatographic procedure as measured by 10 consecutive runs of a single derivatized sample.
b
Precision of the overall derivatization procedure plus chromatographic procedure: 10 amino acid standards were derivatized; one aliquot
of each sample was injected.
c
Internal standard (homoarginine).
d
Hydroxyproline.
e
Hydroxylsine.

ations from the same amino acid standard. The repro-
ducibility of the chromatographic procedure (autosam-
pler, pump, column, and fluorometer) was measured
by 10 consecutive runs of a single derivatized sample.
Table 2 shows the relative standard deviations of reten-
tion time, peak area, and molar response relative to
homoarginine (internal standard) of both procedures.
Both the intra- and interassay variability tests demon-
strate excellent reproducibility for both retention times
and peak areas for all of the amino acids as well as
the peak area relative to the homoarginine internal
standard. Less than 0.1% variation in retention times
is seen in both assays. Multiple injections of the same
derivatized sample showed only 1.6% variation in peak
areas. Peak area variability of the entire procedure (de-
rivatization and chromatography) was as low as 2.0%.
The relatively high variation for Gly (around 4%) is
probably caused by the elution of Gly close to FMOC-
OH; the high variation of Cys is due to the low fluores-
cence response. The inclusion of an internal standard
does not improve the reproducibility, indicating the
high reliability of the assay.
The amino acids Lys, Hyl, and N,O-FMOC-Tyr show
an approximately twofold higher signal than the other
amino acids, due to the presence of a second FMOC
moiety on the e-amino group (Lys, Hyl) and the pheno-
lic hydroxyl group (Tyr) (Figs. 1–3; Table 2). Although
Cys is also a diderivatized amino acid, it shows low
fluorescence properties, presumably due to intramolec-
ular quenching.
Stability of FMOC amino acid derivatives. To es-
tablish that decomposition of FMOC derivatives is ab-
sent with the new protocol, a standard mixture of
amino acids was derivatized at pH 11.4 and injected
immediately. Aliquots of the sample were stored at 47C
and room temperature for 48 h and subsequently ana-
lyzed. Negligible degradation (less than 5%) of the 20
amino acids was observed at both temperatures. At
377C a decrease was seen in homoarginine, Met, Hyl,
and N,O-FMOC-Tyr peak areas. Samples stored over-
night at room temperature showed no decrease in fluo-
rescence response of any of the amino acids, irrespec-
tive if they were stored in daylight or at darkness.
Samples that were not diluted with 25% (v/v) acetoni-

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 9-FLUORENYLMETHYL CHLOROFORMATE AMINO ACID ANALYSIS 173

trile in 0.25 M boric acid after pentane extraction

showed a significant increase (ú15%) of N-FMOC-Tyr
at the expense of N,O-FMOC-Tyr after 7 h at 47C or 3
h at room temperature. After increasing time, degrada-
tion of other amino acids was also observed, resulting
in additional peaks in the chromatogram. This degra-
dation is likely due to the high pH of the derivatized
sample. Dilution of the sample with acetonitrile/boric
acid to lower the pH is therefore a prerequisite to en-
sure the stability of the FMOC derivatives. This dilu-
tion step also improved the chromatography of the
early eluting amino acids, since injection of undiluted
samples caused a broadening of peak width of N-
FMOC-His, Arg, and homoarginine (data not shown).
Recovery of amino acids after extraction with pen-
tane. To check if FMOC derivatives of amino acids
are lost during the extraction with pentane, the recov-
ery was determined after six consecutive extractions.
No differences were observed between samples ex-
tracted once or six times, irrespective of the pH of the
derivatization buffer.
Recoveries of the di-FMOC derivatives of Hyl, Lys, FIG. 4. Effect of evaporation of pentane traces on the adsorption
of amino acids on plastic/glass walls. The amino acid standard was
and Tyr in samples that were not diluted after the
derivatized at pH 11.4 for 40 min. After extraction with pentane the
pentane extraction were highly variable. This was seen cap of the Eppendorf tube was opened for 15 min; the sample was
in samples derivatized in borate buffer at pH 8.0 as applied on the column without diluting it in an organic solvent. Note
well as pH 11.4. This variation was due to variable the high losses of Hyl, Lys, and Tyr (marked with arrows) compared
adsorption of these derivatives on the surface of the with the chromatogram of Fig. 1D. Abbreviations are the same as
in Fig. 1.
polypropylene Eppendorf tubes and the surface of the
glass inserts of the HPLC vials. We speculated that the
presence of residual pentane in the derivatized sample
after extraction prevents the adsorption of disubsti- ratio. The results (Table 3) are in excellent agreement
tuted FMOC amino acids. To substantiate this, evapo- with the expected values. Lysozyme, a protein of 129
ration of pentane traces was stimulated by opening the amino acids with a Mr of 14,000, contains only one His
cap of the Eppendorf tube for 15 min. This resulted in and three Tyr residues. Again, the results, as well as
losses of more than 50% of the diderivatized amino that of albumin and collagen (Table 3), are in good
acids (Fig. 4). The variable adsorption was circum- agreement with the theoretical value, indicating that
vented by diluting the samples with a buffer containing low levels of these amino acids do not lead to erroneous
organic solvent, such as the 25% (v/v) acetonitrile in results.
our studies.
Limit of detection. Because of the high fluorescence DISCUSSION
properties of FMOC derivatives very small amounts The original derivatization procedure of Einarsson
can be detected. Figure 5 depicts the injection of 1 pmol et al. (5) was performed in 0.1 M borate buffer, pH
of each derivatized amino acid (exceptions: homoargi- 7.7, with acetone as solvent. After a reaction time of
nine 2 pmol, Hyp and Pro 5 pmol, Cys 0.5 pmol) at 254/ 40 s at room temperature, excess FMOC-Cl, FMOC-
313 and 254/630 nm. The 630-nm emission shows a OH, and acetone were extracted with pentane. Varia-
significantly lower baseline noise; in this case, the de- tions have been made with respect to the composition
tection limit (signal-to-noise ratio Å 3) is about 200 of the reaction buffer [0.2 M borate (6), 0.5 M borate
fmol for each amino acid. Due to their twofold stronger (16), 0.1 M sodium bicarbonate (10), 0.2 M phosphate
emitting properties the limit of detection of diderivat- buffer (19)], the pH of the reaction buffer [6.3 (12),
ized Hyl, Lys, and Tyr is around 125 fmol. 8.0 (10), 8.5 (19)], the reaction time [1.5 min (19), 3
Analysis of peptide and protein hydrolysates. Hu- min (14), 10 min (10)], the reaction cosolvent [aceto-
man angiotensin-II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) nitrile (19), acetone/acetonitrile (6)], and the extrac-
was used as a model peptide to check the utility of our tion solvent [pentane/ethyl acetate (10)]. Under all
new method on hydrolysates. This peptide (Mr 1047; these reaction conditions, both mono- and di-FMOC
eight amino acids) contains both Tyr and His in a 1:1 derivatives of Tyr are formed, complicating its quan-

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174 BANK ET AL.

FIG. 5. Sensitive detection of a derivatized amino acid standard at two different emission wavelengths. (A) Excitation 254 nm, emission
313 nm; (B) excitation 254 nm, emission 630 nm. Each amino acid represents 1 pmol, except homoarginine (2 pmol), Hyp and Pro (5 pmol),
and Cys (0.50 pmol). Abbreviations are the same as in Fig. 1. Note the high baseline noise at the emission wavelength of 313 nm.

titation. Furthermore, His is always present in the
double-substituted form (FMOC on the a-amino
group and the NH of the imidazole moiety). This ad-
duct shows relatively low fluorescence properties due
to intramolecular quenching and is not stable at room
temperature (it slowly hydrolyzes to N-FMOC-His).
In the derivatization procedure at pH 11.4 for 40
min, His is virtually quantitatively converted to N-
FMOC-His, which is stable at room temperature and
shows a higher fluorescence signal than the disubsti-
tuted form of His. Furthermore, N-FMOC-His elutes
in a clean part, early in the chromatogram (Fig. 1D).
Only small amounts of N-FMOC-Tyr are detectable in
the chromatograms; most of the Tyr is in the form of
N,O-FMOC-Tyr. This derivative has a threefold higher
fluorescence than N-FMOC-Tyr. In addition, its elution
as a free peak at the very end of the chromatogram
further facilitates its quantification.
Two- to threefold improvements for the sensitive de-
tection of all amino acids were achieved by the selection
of a different emission wavelength (630 nm instead of
the widely used 313 nm).
Remarkably, in our method the amount of FMOC-
OH seen in the chromatogram is lower at 40 min
than at 2 min (Figs. 1C and 1D). Since hydrolysis of
FMOC-Cl occurs over time, the opposite was ex-
pected. We have no explanation for this phenomenon.
However, if the derivatization reaction at pH 11.4
was stopped with the hydrophobic 1-adamantylam-
ine and not by extraction with pentane (8, 9, 15, 18),
more FMOC-OH is seen in the chromatograms after
40 min than after 2 min (data not shown). Thus,
somehow the extraction step is involved in the unex-
pected small FMOC-OH peak at 40 min.
The yield of FMOC derivatives has been reported to
be dependent on the concentration of FMOC-Cl to
amino acid (12,16). We have found excellent correlation
coefficients (ú0.999) of calibration curves for molar ra-
tios of FMOC-Cl to total amino acids ranging from 9.6
to 300. This is in agreement with findings of others,
who have shown a linearity of derivatization yield over
a similar large concentration range (17, 19, 20).
The normally used internal standard norleucine or
norvaline interferes with our elution gradient with
other amino acids. It turned out that homoarginine
eluted in a free area, early in the chromatogram (Figs.
1A–1D). Thus, homoarginine can be used in quantita-
tive determinations of unknown samples for the correc-
tion of sample losses during sample handling and prep-
aration and inaccuracies in sample loading onto the
column.
In this paper a ternary gradient was used for amino
acid separation, because of the interest of the authors
in the quantification of Hyl and Hyp in collagen hydro-
lysates. This gradient results in a relatively poor reso-
lution of Ile and Leu. We stress that separation of the
naturally occurring, unmodified amino acids (including
baseline separation of Ile and Leu) can routinely be

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 9-FLUORENYLMETHYL CHLOROFORMATE AMINO ACID ANALYSIS 175

TABLE 3
Amino Acid Composition of Angiotensin-II and Various Proteins Obtained after FMOC-CI Derivatization at pH 11.4

Residues/100 total residuesa

Lysozyme Bovine serum albumin Bovine type II collagen Angiotensin-II

Expectedb Found Expectedb Found Expectedc Found Expectedb Found

His 0.9 1.1 3.1 3.4 0.2 0.3 12.5 12.8

Srg 9.6 9.4 4.2 3.9 5.0 6.1 12.5 12.1
Hyp — — — — 9.7 8.9 — —
Ser 8.7 6.0 4.4 3.4 2.5 2.1 — —
Asx 18.3 20.4 10.3 10.2 4.3 5.4 12.5 13.1
Glx 4.3 5.3 14.4 16.2 8.9 8.6 — —
Thr 6.1 6.0 6.3 5.6 2.3 2.0 — —
Gly 10.4 9.3 2.9 2.4 33.3 33.6 — —
Ala 10.4 9.0 8.5 7.9 10.3 9.5 — —
Pro 1.7 1.9 5.2 5.2 12.0 13.1 12.5 13.0
Met 1.7 0.6 0.7 0.5 1.0 0.4 — —
Val 5.2 5.5 6.6 6.5 1.8 1.8 12.5 11.8
Phe 2.6 2.9 5.0 5.1 1.3 1.3 12.5 12.5
Ile 5.2 6.6 2.6 3.0 0.9 1.1 12.5 12.8
Leu 6.9 7.8 11.2 11.2 2.6 2.4 — —
Hyl — — — — 2.0 1.6 — —
Lys 5.2 5.7 10.9 12.3 1.5 1.4 — —
Tyr 2.6 2.3 3.5 3.2 0.2 0.4 12.5 11.9
a
Proteins were hydrolyzed (200 mg/ml 6 N HCl) at 1107C for 22 h in quadruplicate. After derivatization, aliquots of sample were injected
for HPLC analysis in duplicate. All values are uncorrected for losses during hydrolysis; no attempt was made to determine Cys.
b
Derived from (cDNA) sequence data.
c
Data derived from Miller et al. (11).

achieved with binary gradients (16, 17, 20, 21). No at-
tempt was made to quantitate cysteinyl residues
(which is absent in many collagen chains), which has
low fluorescent properties (Fig. 1 and Table 2). The
cysteine content can best be determined after performic
acid oxidation (converting all cysteine and cystine to
cysteic acid) of the protein before hydrolysis; this deriv-
ative exhibits a response factor similar to other amino
acids and elutes, under the gradient conditions de-
scribed in this paper, in a free area between N-FMOC-
His and Arg (11). Although reduction and subsequent
alkylation of proteins (resulting in S-carboxymethyl-
cysteine) also results in a FMOC derivative with rea-
sonable fluorescence properties, quantification remains
difficult because in the chromatogram it is squeezed
between Thr and Gly (data not shown).
Extraction with pentane or pentane/ethyl acetate
has been reported to result in loss of the hydrophobic
di-FMOC amino acid derivatives (Hyl, Lys, and the
double-labeled derivatives of His and Tyr) (5, 16). We
were not able to reproduce this, but found instead that
significant adsorption of these derivatives occurs on
plastic and glass surfaces when there is little or no
pentane present in the sample. Sometimes losses of
more than 50% or more occurred (Fig. 4). This problem
was easily overcome by diluting the sample with an
organic solvent, such as 25% (v/v) acetonitrile in 0.25
M boric acid.
Injection of pentane-extracted amino acid FMOC
products in the derivatization solution (0.1 M borate,
pH 11.4) onto the HPLC column caused peak-broaden-
ing problems of the first eluting amino acids due to the
high pH. Furthermore, at this high pH a significant
amount of N,O-FMOC-Tyr is converted to N-FMOC-
Tyr within hours at room temperature, followed by deg-
radation of other amino acids. This is due to the fact
that carbamates are not very stable in alkaline solu-
tions, one of the most labile group being the FMOC
substituent on the phenole. Lowering the pH to 9.4 by
dilution of the derivatization mixture with boric acid
overcomes these problems. At this pH, no degradation
is observed for N,O-FMOC-Tyr or other amino acids
within a period of at least 24 h at room temperature
(allowing overnight automated chromatographic analy-
sis) and no chromatographic problems are encountered.
In summary, derivatization of amino acids with
FMOC-OH in 0.1 M borate buffer at pH 11.4 for a pro-
longed period of time (40 min) results in single deriva-
tives of His and Tyr with high fluorescent properties.
The observed decrease in FMOC-OH compared with
short derivatization times results in a more accurate
quantification of Ala. Adsorption of the disubstituted

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176 BANK ET AL.
derivatives after the extraction step is circumvented by
diluting the remaining reaction mixture with organic
solvent. A threefold improvement in sensitivity can be
achieved by the selection of a different emission wave-
length (630 instead of the widely used 313 nm).
ACKNOWLEDGMENT
The work described in this paper was supported by a grant from
‘‘Het Nationaal Reumafonds’’ of The Netherlands.
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