high-density lipoproteins: Comparison of HDL-cholesterol, HDL- density lipoprotein cholesterol, heparin and peripheral vascular

phospholipids, and HDL-apolipoprotein A-I. Clin. Chem. 27,348
(1981). Letter.
8. Telesforo, P., Salino, L., and D’Erico, A., HDL-cholesterol
HDL-phospholipids? Gun. Chem. 27, 354-355 (1981). Letter.
9. Lopes-Virella, M. F., Stone, P., Ellis, S., and Caidwell, J. A., Cho-
lesterol determination in high-density lipoproteins separated by three
different methods. Clin. Chem. 23,882-884 (1977).
10. Alcindor, L. G., Dusser, A., Piot, M. V., et al., A rapid method for
lecithin:cholesterol acyltransferase estimation in human serum.
Scand. J. Clin. Lab. Invest. 38, Suppi. 150, 12-15 (1978).
11. Folch, J., Lees, M., and Stanley, G. H. S., A simple method for the
isolation and purification of total lipids from animal tissues. J. Biol.
Chem. 226, 497-509 (1957).
12. Masana, L., Lagtma, J., Masdeu, S., et al., High density lipoprotein
cholesterol and coronary heart disease. In Proceedings of Florence
disease. Biomedicine 35,42-43 (1981).
15. Vergani, C., Trovato, G., DelO, A., et al,, Serum total lipids, Ii-
or poprotein cholesterol, and apolipoprotein A in acute viral hepatitis
and chronic liver disease. J. Clin. Pathol. 31, 772-778 (1978).
16.Yamamoto, K.,Koga, S.,and Ibayashi, H., Apoprotein A-I in
cholestatic liver disease. Clin. Chim. Acta 87, 85-92 (1978).
17. Gjone, E., and Norum, K. R., Plasma lecithin:cholesterol acyl-
transferase and erythrocyte lipids in liver disease. Acta Med. Scand.
187, 153-161 (1970).
18. Simon, J. B., and Scheig, R., Serum cholesterol esterification in
liver disease: Importance of lecithin:cholesterol acyltransferase. N.
Engi. J. Med. 283,841-846 (1970).
19. Calandra, S., Martin, M. J., and McIntyre, N., Plasma lecithin:
cholesterol acyltransferase activity in liver disease. Eur. J. Gun. In-
vest. 1, 352-360 (1971).

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International Meeting on Myocardial Infarction, D. T. Mason, C. 20. Rubi#{233}s-Prat,J., Ras, M. R., Frisbn, J. C., et al., Effect of heparin
G. Neri-Serneri, and M. F. Oliver, Eds., Excerpta Medica, Amsterdam, on lecithin:cholesterol acyltransferase and lipids in cholestasis. Di-
1979, pp 433-435. gestion 14, 250-255 (1976).
13. Nubiola, A. R., Masana, L., Masdeu, S., and Rubibs-Prat, J., High 21. Harry, D. S., Day, R. C., Owen, J. S., et al., Plasma lecithin:cho-
density lipoprotein cholesterol in cerebrovascular disease. Arch. lesterol acyltransferase activity and the lipoprotein abnormalities of
Neurol. 38, 468 (1981). liver disease. Scand. J. GUn. Lab. Invest. 38,Suppl. 150, 223-227
14. Masana, L., Rubi#{233}s-Prat, J., Nubiola, A. R., et al., Serum high- (1978).

CLIN. CHEM. 28/3, 527-531 (1982)

Rapid Assay for Amino Acids in Serum or Urine by Pre-Column

Derivatization and Reversed-Phase Liquid Chromatography
D. C. Turnell and J. D. H. Cooper

This method for estimating clinically important amino acids We describea rapid method forthe quantitativeestimation
in serum or urine within 40 mm involves o-phthalal- of clinically
important freeamino acidsin serum or urine,by
dehyde/2-mercaptoethanol derivatization and reversed- using fluorescencedetectionof OPA/MCE derivatives of the
phase “high-pressure” liquid chromatography. Homo- analytes,and theirseparation by gradient elutionreversed-
cysteic acid is an internal standard, and homoserine and phase HPLC.
norvaline are reference peaks. For all the amino acids
estimated, the between-run coefficients of variation ranged
from 2.0 to 13.5%, and the mean analytical recoveries Materials and Methods
from bothserum and urine samples was 101%. Peak Instrumentation
areas vary linearly with concentration up to 1500 umoi/L
for all the amino acids assayed. The limit of detection for The gradient HPLC system used was an Altex 420 (Altex
ScientificInc.,Berkeley, CA 94710). Injectionswere made
each amino acid was estimated to be 38 fmol.
using a Rheodyne valve fittedwith a 20-zL loop.The amino
The quantitativeestimation of freeamino acids by “high- acid derivativeswere detected with a Schoeffel FS 970 Fluo-
rescence Detector (Kratos Inc.,Westwood, NJ 07675), exci-
pressure”liquidchromatography (HPLC) ispotentiallyfaster
tationwavelength 230 nm and emissioncutofffilter at 418 nm.
and more sensitive(1, 2) than the classicalion-exchange
The data from the chromatography was processed by a
methods (3).
SP4100 Computing Integrator (Spectra-Physics,Santa Clara,
Pre-column o -phthalaldehyde/2-mercaptoethanol (OPAl
CA 95051). We used a 150 X 4.6 mm i.d.analyticalcolumn
MCE) derivatization is ideally suited to amino acid analysis
pre-packed with 5-tim diameter Ultrasphere ODS (Altex
by reversed-phase chromatography because the derivatives
produced are lesspolar than the freeamino acids.Moreover, Scientific Inc.). The analytical column was fittedwith a 70 X
2 mini.d. pre-column packed with 25- to 37-mm diameter
the formation of these amino acid derivativesfrom physio-
logical fluids is rapid and can be performed in aqueous con- CO:PELL ODS (Whatman Inc.,Clifton,NJ 07014).
ditions. The problem of poor fluorescence intensity for the Reagents
cystine derivative is overcome by pretreating the samples with
All amino acids were obtained from the Sigma Chemical
iodoacetic acid (4).
Co., St. Louis, MO 63178, lodoacetic acid and 2-mercapto-
ethanol were purchased from Aldrich Chemical Co., Mil-
Biochemistry Department, Coventry and Warwickshire Hospital, waukee, WI 53233. “Far UV-grade” acetonitrile was obtained
Stoney Stanton Rd., Coventry CV1 4FH, U.K. from Fisons Scientific
Apparatus, Loughborough, U.K. Unless
Received Nov. 9, 1981, accepted Dec. 16, 1981. stated,allother chemicals were analyticalgrade, obtained

CLINICALCHEMISTRY,Vol. 28, No. 3, 1982 527

 and 0.62 g of boric acid in approximately 50 mL of water.
Table 1. StabilIty of Amino Acid OPA/MCE Adjust topH 9.5with 2 mollL sodium hydroxide solutionand
Derivatives in Two Different Solvents diluteto 100 mL with water.
OPA/MCE derivatIve Acetonitrile precipitation reagent: Add 200 iL of 2-mer-
half-life, mln captoethanol to 100 mL of acetonitrile.Prepare thisreagent
DIluted In Diluted In daily.
Amino acId OPA/MCE reagent enivent A
Phosphoserine 3.1 2.3 ChromatographiC Conditions
Aspartic acid 7.4 13.9 Filter all the HPLC solvents through a 0.5-tm pore size
Glutamic acid 15.5 10.9 filter (Millipore Intertech Inc., Bedford, MA 01730) and de-
Homocysteic acid 6.7 92.8 gas by bubbling with helium before use. Prepare a stock so-
Cystine8 dium propionate solution containing, per liter, 250 mmol of
8.6 25.4
propionic acid (relative density = 0.990-0.995 kg/L), and 350
a-Aminoadiplc acid 13.6 30.8
mmol ofanhydrous disodium hydrogen phosphate. Adjust this
Asparagine 10.2 16.0 solution to pH 6.50 (±0.01) at room temperature with sodium
Homocystine8 6.7 20.8

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hydroxide, 4 mol/L.
Serine 11.6 Solvent A: water/stock sodium propionate solutionlace-
Histidine 44.5 21.3 tonitrile (72/20/8 by vol).
Glutamine 20.3 21.8 Solvent B: waterlacetonitrile/methanol/dimethyl sulfoxide
(42/30/25/3by vol).The gradient program used isshown in
Ethanolamine 0.7 2.6
the recorder trace (Figure 1).
phosphoric acid
Homoserine 33.0 13.1 Procedures
Glycine 0.9 All standards, quality-assessment specimens, and test
Threonine 26.0 30.4 specimens were stored at -20 #{176}C and thawed immediately
Citrulline 3.5 79.3 before analysis.
Arginine 43.0 18.4 Derivatization: To 20 iL of standard amino acid solution,
3-Methyihistidine serum, or urine in a polypropylene tube add 20 L of internal
29.9 6.8
standard solutionand 200 L ofthe acetonitrile precipitation
f3-Alanmne 2.7 6.4 reagent.Vortex-mix the contents of alltubes and centrifuge
Alanine 9.4 15.6 at 12 000 X g for2 mm. Take 20 iL ofthe supernatant solution
Taurine 1.7 13.2 into another polypropylene tube and add 100 tL of the io-
Tyrosmne 37.2 39.3 doaceticacid reagent.Mix the contents of the tube, add 100
ct-Amino-n-butyric 27.3 16.4 L of the OPA/MCE reagent,and without delay inject20 tL
acid onto the column.
Quantitation: Amino acidderivatives are identified by their
Ethanolamine 2.9 6.9
retentiontimes relativeto the referencepeaks produced by
Valine 23.1 49.4 homocysteic acid,norvaline,and homoserine. The amino acid
Methionine 29.2 23.7 concentrationsare quantifiedby comparing theirpeak areas
Norvaline 33.6 with that of homocysteic acid,the internalstandard.Quality
Tryptophan 20.2 62.4 of the analyticaldata was assessed by assaying in every run
Phenylalanine 22.6 48.0 two serum and two urine samples that had been supplemented
lsoleucine 43.5 48.9 with appropriate amino acids to yield a high and low con-
centration of analytes in each material.
Leucine 21.5 35.3
Stability of the amino acid OPA/MCE derivatives: The
Hydroxylysmne 2.4 29.1
stability of the amino acid OPA/MCE derivatives was exam-
Ornithine 1.9 72.3 ined in two ways:
Lysine 3.7 31.4 1. To 200 zL of a 100 mol/L amino acid solution add 2.0
Ammonia 1.4 15.6 mL of OPA/MCE reagent and monitor continuously the flu-
8 Estimated as a derivative of iodoacetlc acid. orescence produced for 30 mm. We used a Gilson Spectra-Gb
fluorometer (Anachem Ltd., Luton, U.K.) with 360 nm exci-
tation and 455 nm emission filters.
2. To establish the stability of the amino acid derivatives
from BDH Chemicals Ltd., Poole, U.K. Distilledde-ionized in solvent A, add 200 sL of a 1.0 mmol/L amino acid solution
water was used forallreagent preparations. to 2.0 mL of the OPA/MCE reagent. Immediately dilute 20
OPA/MCE reagent: Dissolve 500 mg of OPA (Sepramar tL of thismixture with 2.0mL of solvent A and monitor the
grade,BDH Chemicals Ltd.) in 10 mL of methanol and dilute fluorescenceas described previously.
to 100 mL with a 400 mmol/’L sodium borate buffersolution
(pH 9.5).To thissolutionadd 400 iL of 2-mercaptoethanol Results
followedby a further40 tL every three days (2). This reagent Derivatization: The derivatizationmethod described is
was stableindefinitelyat room temperature. based on one we developed earlier(4),which overcame the
Standard amino acid solution: Dissolve in 1.0L of water problem of poor fluorescenceof the cysteineOPA/MCE de-
500 mol of each amino acid listedinTable 1.Store aliquots rivative and used perchloric acid precipitation for sample
of thisstandard solutionat -20 #{176}C. preparation. Reduced sensitivity of lysine, hydroxylysine, and
Internal standard solution: Dissolve in 1.0L of water 1,0 particularly of ornithine observed with this method (4) is re-
mmol of the following amino acids:homocysteic acid,ho- stored by substituting acetonitrile for perchloric acid pre-
moserine,and norvaline.Store aliquotsof this solutionat -20 cipitation. As can be seen in Figure la, the use of iodoacetic
#{176}C. acid to overcome the cysteine problem (4) operates equally
lodoacetic acid reagent: Dissolve0.74 g of iodoaceticacid wellforhomocysteine.

528 CLINICAL CHEMISTRY, Vol. 28, No. 3, 1982

 I0
I.

I
0

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0 10 20 30 40

T (tINS)
(a)
10 100
lb

181
a

I.’
50

28

10
4

20
0 lb 30 40
TIME I U INS
(b)
100 100
lc

‘SI a
S

50
,,

n __________________________________________________________
0 10 20
______________________
40
TIME CUINS)
(c)
FIg. 1. Amino acid chromatograms of (a) 500 moI/L standard solution, (b) adult serum, and (C) random (untimed) adult urine
sample
The standard and tests were prepared as described in the text. The amIno acid peaks are numbered as in Table 2. IS1 = homocysteic acid, 1S2 homoserine,
1S3 = novallne. &oken lines indicate solvent gradient composition

CLINICAL CHEMISTRY, Vol. 28, No. 3, 1982 529

 Table 2. Within-Run and Between-Run PrecIsion of Amino Acid Estimation
WIthin-run (n 10) CV, %:
Concn In sample, igmol/L Between-run (n = 10) CV, %
5-20 21-200 201-1000 (and mean, pmoi/L)
Amino acId 8 Serum Urine Serum Urine Serum Urine Serum Urine
1. Phosphoserine b 4.2 b 2.4 b 2.1 b 7.9 (12)
2. Aspartic acid 8.8 2.7 1.7 2.4 b 1.4 4.8(40) 8.5(13)
3. Glutamic acid 5.3 2.8 3.3 1.6 0.8 b 5.4 (103) 2.6(83)
4. Cystinec 4.1 5.0 2.3 2.3 1.2 1.2 4.2(114) 2.5(263)
5. a-Aminoadipic acid b 10.2 b 99 b 1.6 b 13.5 (70)
6. Asparaglne 9.0 8.4 1.2 4.8 1.3 4.6 2.6(58) 8.9(107)
7. Homocystine b 9.1 b 6.9 b 39 b 9.8(49)
8. Serine 5.3 6.1 5.1 4.4 1.6 1.0 6.8 (144) 1.8(286)
9. Histidine 12.6 8.4 2.0 4.9 2.4 1.1 5.1 (150) 2.1 (615)

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10. Glutamine 5.7 6.2 3.5 3.2 1.1 1.1 2.1 (882) 2.1 (554)
b b
11. Ethanolamine phosphoric acid 13.5 9.8 b 5.1 b 44
12. Glycine 5.8 6.4 4.9 3.3 2.8 1.1 5.4 (270) 2.0 (936)
13. Threonine 5.7 5.6 3.2 1.8 0.8 0.8 3.5 (190) 2.5 (510)
14. Citrulline 10.5 7.4 2.1 4.7 1.4 b 4.2 (25) b

15. Arginine 6.3 5.3 2.4 1.6 1.7 1.6 4.4(108) 2.6 (1 19)
16. 3-Methylhistidine b 5.1 b 1.5 b 1.2 b b

17. f3-Alanine b 6.5 b 1.6 b b b 3.2 (33)

18. Alanine 5.5 6.0 3.8 2.8 1.5 1.1 3.1 (353) 3.8 (187)
19. Taurine 6.4 5.2 3.5 3.1 2.0 1.4 5.7 (86) 2.8 (410)
20. Tyrosine 9.8 6.6 1.5 1.9 1.3 1.1 2.9(82) 4.1 (132)
21. a-Amino-n-butyric acid 6.6 8.9 7.4 3.4 2.8 2.2 11.3 (17) 11.8 (14)
22. Ethanolamine 7.0 7.3 b 35 b 2.0 12.3 (7) 3.0 (305)
23. Valine 6.5 13.4 2.6 1.6 1.2 b 2.1 (249) 2.5 (43)
24. Methionine 8.9 8.8 2.4 4.8 2.3 b 11.2(19) 12.4(8)
25. Tryptophan 10.2 10.7 2.0 3.8 b 4.5 2.8 (50) 5.9(137)
26. Phenylalanine 6.3 6.4 1.8 1.6 1.0 1.4 3.5 (95) 3.3 (67)
27. Isoleucine 9.2 2.5 1.8 4.8 1.6 b 3.4(74) 5.3(14)
28. Leucine 7.1 8.4 3.2 3.7 0.7 1.0 5.8 (130) 2.5 (537)
29. Hydroxylysine 12.9 6.6 b 3.1 b 7.2 b 5.4 (87)
30. Ornithine 8.1 7.9 1.1 5.1 2.9 5.5 2.1(81) 8.3(61)
31. Lysine 11.2 8.1 5.2 4.4 1.5 1.7 8.3 (164) 2.8 (577)
a Numbers correspond to numbered peaks In FIgure 1. b Amino acid not detected. C Estimated as a derivative of iodoacetic acid.

Stability of derivatives: The half-lives of a range of amino fluorescenceratioswere obtained from chromatography of 40
acid OPA/MCE derivativesin the OPA/MCE reagent and differentserum and urine specimens.
after diluting in solvent A are shown in Table 1. The mean analytical recovery was 101% (SD 2.3%), as esti-
Chromatography: The amino acidchromatograms of a pure mated from analysis of serum and urine samples supple-
standard and of serum and urinesamples areshown in Figure mented with known quantities of amino acids.
1. Chromatographic stabilitywas good, the peak retention Under the conditions described, the peak areas are linear
times, either between runs or between changes of column, with concentration up to 1500 tmol/L foreach amino acid.
varying lessthan 10% when ambient temperatures were kept Sensitivity: Using the fluorometer setting and chromato-
constant (±2 #{176}C). This variation never reduced the resolution graphic conditions described but with the integratorpeak
of the analytes. threshold set at one (usuallyset at 12) we analyzed a zero
Precision: To estimate the within-run precisionat three standard. Comparison of the peak areas that resultedfrom
concentrations of analyte,we assayed differentvolumes of baselinenoisewith that of the internal standard allowed cal-
human serum and urine samples in replicate(Table 2).Be- culation of the limit of detection. The limit of detection for
tween-run precision was estimated by analyzing aliquots of each amino acid on-column was 38 fmol, estimated as the
human serum and urineon 10 differentdays.Aliquotsof each mean +2 SD concentration of the zero standard peaks (n =
sample were storedat -20 #{176}C and thawed justbeforeanalysis 105).
(Table 2).
Accuracy: The standard amino acid solution was chroma- Discussion
tographed afterthe derivatizationprocedure at fluorometer The stability of OPA/MCE amino derivatives in OPA/MCE
excitationwavelengths of 230 and 330 nm, the two peak reagent (Table 1) was similar to those previously observed by
maxima forexcitationof OPA/MCE derivatives.After using Lindroth and Mopper (2). To estimate the decay of the de-
the internal standard to correct for injection variations, we rivatives during the first part of the chromatographic process,
calculated the ratio of the fluorescence produced at these we diluted them in solvent A and estimated theirhalf lives
wavelengths for each amino acid. Except for taurine, the same (Table 1). The general increase in stability is probably due to

530 CLINICAL CHEMISTRY, Vol. 28, No. 3, 1982

the organic content of the matrix (5). The interference of
ammonia with amino acid estimations in ion-exchange chro-
matography dose not occur with this method because free
ammonia, contaminating eluent solvents,does not adsorb to
reversed-phase columns and its derivativewith OPA/MCE
has a low fluorescenceintensityand shorthalf-life
compared
with that of the amino acids (Table 1). In spite of the varia-
tions in stabilities of the derivatives, if care is taken to re-
produce the time between derivatization and injection, good
overall analytical precisions are obtained (Table 2).
The fluorescence response ratios obtained for the standards
and tests suggest that, except for taurine, there is no inter-
ference with the estimation of the amino acids assayed. The
interference with taurine estimation is probably caused by
$-aminoisobutyric acid.
When chromatographed, D,L-isoleucine produced a fused
double peak. Using L-isoleucine in the standard produced a
single peak; this phenomenon was not observed for any other
amino acids investigated.
Propionic acid is included in solvent A to obtain good peak
symmetry for 3-methyihistidine. The use of methanol alone
as the organic solvent gave excellent peak shape and resolution
for most of the amino acids but, to resolve glycine and threo-
nine, acetomtrile had to be substituted for methanol in solvent
A. The mechanism of this separation is probably adsorption
of threonine onto free silanol groups, which is enhanced by the
omission of alcohol from the mobile phase (6). The addition
of a small amount of propionate solution in solvent B en-
hanced the resolution of valine and methionine.
Because the concentrations of individual serum and urine
amino acids vary widely, good chromatographic resolution is
required for the quantitation of analytes. An example of this
is the separation of glycine and threonine, the molar ratio of
which is approximately 20:1 in normal urine (Figure ic). We
chose a standard solution with a concentration of 500 mol/L
for each amino acid; to encompass this wide variation. To
minimize the volume of sample required, smaller volumes may
be used in the pretreatment procedure, or the fluorometer
sensitivity may be increased.
Compared with dedicated amino acid analyzers based on
ion-exchange chromatography, this HPLC method is faster,
more sensitive, and cheaper to operate. Furthermore, because
of the analytical versatility of HPLC, the same instrumenta-
tion used for amino acid analysis may be used for many other
assays. Although the method described requires manual
sample preparation and injection, the system has the potential
of offering fully automated chromatographic analysis of amino
acids in physiological fluids.
We thank Mrs. C. Fuller, Miss N. Shearsby, and Mr. M. Lewis for
their excellent technical assistance, and Anachem Ltd., Luton, U.K.,
for the loan oftheGilsonSpectra-Gb fluorometgr.
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References
1. Hill, D. W., Walters, F.H.,Wilson,T. D.,and Stuart,J.D.,High
performanceliquidchromatographicdeterminationofamino acids
inthe picomoterange.Anal. Chem. 51, 1338-1341 (1979).
2. Lindroth, P., and Mopper, K., High performance liquid chroma-
tographic determination of subpicomole amounts ofamino acids by
precolumn fluorescence derivatization with o-phthalaldehyde. Anal.
Chem. 51, 1667-1674 (1979).
3. Moore, S., Spackman, D. H., and Stein, W. H., Chromatography
of amino acids on sulfonated polystyrene resins. Anal. Chem. 30,
1185-1190(1958).
4. Cooper, J. D. H., and Turnell, D. C., The fluorescence detection
of cystine by o-phthalaldehyde derivatization and its separation using
high performance liquid chromatography. J. Chromatogr. 227,
158-161(1982).
5. Simons,S.S.,and Johnson, D. F., Reaction of o-phthalaldehyde
and thiols with primary amines: Fluorescence properties of 1-alkyl
(and aryl) thio-2-alkylisoindoles. Anal. Biochem. 90, 705-725
(1978).
6. Martin, A. J. P., and Synge, R. L. M., A new form of chromatogram
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