3740
`
`Chern. Pharm. Bull.]
`35( 9 )3740-3745(1987)
`
`[
`
`Vol. 35 (1987)
`
`Determination of Free and Total Phenylacetic Acid in Human and Rat
`Plasma by High-Performance Liquid Chromatography
`with Fluorescence Detection
`
`MASATOSHI YAMAGUCHI* and MASARU NAKAMURA
`
`Faculty of Pharmaceutical Sciences. Fukuoka University,
`Nanakuma. Johnan-ku, Fukuoka 8/4-01, Japan
`
`(Received February 9, 1987)
`
`A highly sensitive and simple high-performance liquid chromatographic method has been
`developed for the determination of free and total phenylacetic acid in human and rat plasma. After
`extraction with diethyl ether from plasma, phenylacetic acid and phenylpropionic acid (internal
`standard) are converted to the corresponding fluorescent derivatives by reaction with 3-
`bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydro(cid:173)
`gen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase
`column (Radial-Pak cartridge C 18 ) with aqueous 65% (v/v) methanol and detected fluorimetrically.
`The detection limit for phenylacetic acid is II pmol(ml in plasma at a signal-to-noise ratio of 5. This
`sensitivity permits precise determination of free and total phenylacetic acid in 50111 of human and
`rat plasma. The method was applied to the determination of free and total phenylacetic acid in
`plasma from healthy volunteers, and control and "behavioral despair" rats.
`
`Keywords----phenylacetic acid; human plasma; rat plasma; high-performance liquid chroma(cid:173)
`tography; fluorescence detection; 3-bromomethyl-6, 7-dimethoxy-1-methyl-2( I H )-quinoxalinone
`
`>
`
`Phenylacetic acid (P AA) is present as free and conjugated forms in human plasma. P AA
`may be mainly derived from phenylalanine and phenylethylamine by decarboxylation and
`deamination, and is further metabolized in the human body to its glutamine conjugate. It is
`indicated that the amount of PAA decreases in plasma of patients with depressive illness. 1
`2
`•
`Therefore, the determination of plasma PAA in humans may be useful for the diagnosis,
`monitoring and investigation of depressive illness.
`Recently, rats forced to swim inJl restricted space have been found to be a specific animal
`model for depressive illne"ss. Thus, the determination of plasma PAA in such rats may improve
`our understanding of depressive illness. 3 )
`Gas chromatography-mass spectrometric (GC-MS) methods have been most widely used
`for the determination of free and total (the sum of free and conjugated) PAA in human
`4
`7
`plasma. 2
`) Although the methods are very sensitive, they require expensive equipment and
`•
`-
`rather tedious techniques. Thus, they have not been routinely used. Recently, a simple high(cid:173)
`performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection has
`been proposed for the determination of total PAA in human plasma.!) However, the method
`has a limited sensitivity and thus requires a large amount of human plasma (2 ml).
`Furthermore, the method has not been applied to the determination of free P AA, which
`occurs in a minute amount in human plasma. No method has yet been applied to experimental
`small animals such as rats and mice.
`We previously reported a simple and sensitive HPLC method for the simultaneous
`determination of free and total PAA and p- and m-hydroxyphenylacetic acids in human urine
`using precolumn fluorescence derivatization with 3-bromomethyl-6,7-dimethoxy-l-methyl-
`2(1 H)-quinoxalinone (Br-DMEQ), a fluorogenic reagent for carboxylic acids; these acids,
`
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`

`

`No.9
`
`3741
`
`after extraction with diethyl ether from urine, are converted into the corresponding
`fluorescent compounds by reaction with Br-DMEQ and these compounds are separated on a
`reversed-phase (Radial Pak cartridge C 18) column with isocratic elution. 8 l The purpose of the
`present research was to establish a simple, sensitive and rapid method for the determination of
`free and total PAA in a minute amount of human and rat plasma. The established method
`was used to compare free and total PAA concentrations in control rat plasma with those in
`plasma from rats forced to swim. Phenylpropionic acid (PPA), which is not present in human
`and rat physiological fluids, was used as an internal standard (IS).
`
`Experimental
`
`Chemicals and Solutions~-All chemicals and solvents were of reagent grade, unless otherwise stated. Deionized
`and distilled water was used. PAA was purchased from Sigma (St. Louis, Mo., U.S.A.). Acetonitrile used for the
`derivatization reaction was purified as described previously.9 l Br-DMEQ was prepared as described previously;9 l it is
`now available from Dojindo Laboratories (Kumamoto, Japan). Br-D).·IEQ (1.3 mM)'; 18-crown-6 (3.8 mM) and PPA
`(3.2,uM, IS) solutions were prepared in acetonitrile. The Br-DMEQ solution could be kept for more than one week
`when stored in a refrigerator at 4 oc.
`Apparatus and HPLC Conditions~-A Hitachi 655A high-performance liquid chromatograph equipped with a
`high-pressure sample injector and a Hitachi FIOOO fluorescence spectromonitor equipped with a 12-,ul flow-cell
`operating at the excitation and emission wavelengths of 379 and 455 nm, respectively, were used. The column was a
`Radial Pak cartridge C 18 ( 100 x 8 mm i.d.; particle size, 5,um; Waters Assoc., Milford, Mass., U.S.A.). The mobile
`phase was H20-MeOH (35: 65, vjv). The flow-rate was 2.0 mljmin (ca. 70 kgjcm2
`). The column temperature was
`maintained at 40 ± I oc. This column could be used for more than 1000 injections with only a small decrease in the
`theoretical plate number when washed with methanol at a flow rate of 2 ml/min for ca. 20 min at the end of each
`working day. Uncorrected fluorescence excitation and emission spectra of the eluates were measured with a Hitachi
`650-60 fluorescence spectrophotometer fitted with a 20-,ul flow-cell; the spectral bandwidths were 5 nm in both the
`excitation and emission monochromators.
`Plasma Samples--Male Wister rats (n = 20) weighing 210-230 g were used for the present study. The rats were
`housed in a well-controlled environment with free access to food and water, and were used within a day after being
`brought into the laboratory. The rats were divided into two groups of 10 rats each. One group (intact rats) served as a
`control. The other group (10 rats) was treated according to the novel forced swimming test of Porsolt et a[. 3l Briefly,
`the rats were individually forced to swim once daily inside plexiglass cylinders containing water maintained at 25 oc
`for 15 min. After 4 daily sessions of swimming, the total duration of immobility was measured during a 5 min test. All
`the rats employed were judged from the forced swimming test to be in a depressive state. On the next day, the rats
`were killed by decapitation, and blood (2--4 ml) was collected in a centrifuge tude containing disodium
`ethylenediaminetetraacetate (2--4 mg) as an anticoagulant. Plasma was separated by centrifugation of the blood at
`10000 g at 5 oc. Human plasma was obtained from fasting healthy volunteers in our laboratory. Human and rat
`plasma samples were stored at -40 oc until just before use.
`Procedure~-A 50-,ul portion of plasma sample was diluted with 50 ,ul of the PPA (IS) solution, I 00 ,ul of 0.2 M
`zinc sulfate, and 100 ,ul of 0.2 M barium hydroxide. The mixture was centrifuged at 6500 g for 20 min. The supernatant
`(deproteinized plasma; 200 ,ul) was mixed with 50 ,ul of 6 M hydrochloric acid, and the acidified plasma was hydrolyzed
`at 100oc for 90 min. To the resulting solution, 2 ml of diethyl ether was added, and the resulting mixture was vortexed
`for ca. 2 min and centrifuged at 1000 g for 2 min. The organic layer (ca. 1.4 ml) was evaporated to dryness in vacuo at
`15-20 oc and the residue was dissolved in 200 ,ul of acetonitrile. A I 00-,ul portion of the final solution was placed in
`a screw-capped 10-ml vial, to which were added ca. 20mg of a mixture of potassium hydrogen carbonate and
`potassium sulfate (I :4, w/w) and 50 ,ui each of the Br-DMEQ and 18-crown-6 solutions. The vial was tightly closed
`and warmed at 50 oc for 30 min in the dark. After cooling, 20 ,ul of the resulting mixture was injected into the
`chromatograph. For the determination of free PAA, the same procedure was carried out except that hydrolysis was
`omitted.
`The calibration graphs were prepared according to the standard procedure except that 50 ,ui of the PPA (IS)
`solution was replaced with the IS solution containing 50pmol-100nmol ofPAA. The net peak height ratio ofPAA
`was plotted against the concentration of PAA spiked.
`
`Results and Discussion
`
`HPLC and derivatization conditions were the same as described previously. 8 l
`
`Deproteinization
`Plasma had to be deproteinized, otherwise the HPLC column packing was considerably
`
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`

`

`3742
`
`500
`
`A
`
`B
`
`Vol. 35 (1987)
`
`0
`
`0
`
`30
`
`60
`Time (min)
`
`90
`
`Fig. I. Effect of the Reaction Time on the
`Hydrolysis of Conjugated PAA in (A) Human
`and (B) Control Rat Plasma
`
`120
`
`Portions (50 ,ul) of the plasma were treated accord(cid:173)
`ing to the standard procedure.
`
`damaged. The deproteinization was effectively done by adding zinc sulfate and barium
`hydroxide to plasma. When plasma was deproteinized with perchloric acid, an unknown,
`broad and large peak appeared at the retention time of 10-30min on the chromatogram.
`
`Hydrolysis
`The optimal conditions for hydrolysis of conjugated P AA were examined by using
`pooled human and rat plasma. When the deproteinized plasma was acidified with an equal
`volume of 6 M hydrochloric acid, and hydrolyzed at 100 oc for 60-120 min, the conjugated
`PAA in human and rat plasma was almost completely hydrolyzed, as shown in Fig. I. Thus,
`the acidified plasma was heated at 100 oc for 90 min in the procedure for the determination of
`total PAA.
`
`Extraction
`PAA was effectively extracted from the acidified plasma before and after hydrolysis with
`diethyl ether. A recovery test was performed by adding a known amount (50 pmol) ofPAA to
`human plasma (SO,ul). Recovery ofPAA was 50.2±3.2% (mean±S.D., n= 10). Main loss in
`PAA occurred in the protein precipitation step. Less satisfactory recoveries were found with
`ethyl acetate, benzene and chloroform. Similar results were also obtained for rat plasma.
`
`Chromatography
`Figure 2 shows a typical chromtltogram obtained with a standard mixture ofPAA,p- and
`m-hydroxyphenylacetic acids and PPA. 8
`> The peaks for the acids (peaks 2-5) could be
`completely separated from the components of the reagent blank (Fig. 2, peaks 6 and 7) within
`26 min. Figures 3A and B show typical chromatograms obtained with pooled human and rat
`plasma, respectively, before and after hydrolysis. The component of peak 2 was identified as
`the DMEQ derivative of PAA on the basis of the retention time and the fluorescence
`excitation (maximum, 370 nm) and emission (maximum, 455 nm) spectra of the peak fraction
`by comparison with those in Fig. 2, and also by co-chromatography of the standard
`compound and plasma with aqueous 50-100% methanol as the mobile phase. On the other
`hand, no peaks for p- and m-hydroxyphenylacetic acids were observed in the chromatograms,
`because the acids occur in extremely small amounts in human plasma. 2• 5 · 7 l Some unidentified
`peaks (Fig. 3A and B, peaks 8-1 0) were observed on the chromatogram. The heights of these
`peaks increased in proportion to the plasma sample size. In addition, each eluate from peaks
`8--10 exhibited fluorescence excitation and emission maxima around 370 and 455 nm almost
`identical with those of peaks 2-5 (Fig. 2). These observations suggest that peaks 8~10 may
`be due to unknown endogeneous carboxylic acids in plasma. However, they did not interfere
`with the determination of PAA in plasma. No conversions of phenylacetaldehyde and
`
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`

`

`No.9
`
`3743
`
`<BJ
`
`16
`Time (min)
`
`Fig. 2. Chromatogram of the DMEQ Deriva(cid:173)
`tives of PAA, p- and m-Hydroxyphenylacetic
`Acids and PP A
`A portion (50 pi) of a standard mixture of the acids
`(lOnmol each/ml) in water was treated according to
`the standard procedure. Peaks: I, Br-DMEQ; 2, PAA;
`3, m-hydroxyphenylacetic acid; 4, PPA; 5, p-hy(cid:173)
`droxyphenylacetic acid; 6 and 7, the reagent blank.
`
`16
`Time <minl
`
`Time (min>
`
`(A)
`Fig. 3. Chromatograms Obtained with
`Healthy Human and (B) Control Rat Plasma
`(--) before and (------)after Hydrolysis
`Experimental details are described in the text. For
`peaks 1-7. see Fig. 2; peaks 8-10 are unidentified.
`
`phenylpyruvic acid, which occur in biological fluids, to PAA during the procedure were
`observed even when they were present at unusually high concentrations in plasma
`(5.0 nmol/ml in plasma). Thus, further clean-up of the sample solution was not necessary.
`Linearity, Detection Limit and Precision
`A linear relationship was observed between the ratio of the peak height ofPAA to that of
`PPA and the amounts of PAA (50pmol-100nmol) added to 50J1l of human plasma. The
`linear regression equation (the linear correlation coefficient in parenthesis) was Y =
`0.02053X +0.0022 (r=0.998), where Y and X are the peak height ratio and the concentra(cid:173)
`tion (nmoljml) of PAA, respectively.
`The detection limit for PAA was 11 pmoljml in plasma at a signal-to-noise ratio of 5. The
`sensitivity is much higher than that of the UV-HPLC method, and is comparable to those of
`the GC-MS methods.
`The within-day precision was determined from repeated analyses (n 20) of a normal
`human plasma containing 0.35nmol/ml of free PAA and 2.93nmol/ml of total PAA. The
`coefficients of variation were 3.9 and 3.6% for free and total PAA, respectively. The between(cid:173)
`day precision was obtained by performing the analyses (n = 3 each day) using the calibration
`graph prepared on that day during ten days with plasma samples kept frozen at -40 oc_ The
`coefficients of variation were 4.1 and 4.3% for free and total PAA, respectively.
`
`P AA Concentration in Human Plasma
`The levels of free, total and conjugated (calculated from differences) PAA in human
`plasma are given in Table I. The mean values were in agreement with those obtained by other
`
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`

`

`3744
`
`Vol. 35 (1987)
`
`TABLE I. Plasma Concentrations ofPAA (nmol/ml) in Normal Human
`
`Age
`
`Sex"'
`
`Free
`
`Conjugate•>
`
`Total
`
`Free/total
`(mol( mol)
`
`M
`M
`M
`M
`M
`M
`M
`M
`M
`M
`
`F
`F
`F
`F
`F
`F
`
`59
`37
`35
`31
`28
`27
`27
`24
`24
`23
`Mean
`S.D.
`
`25
`25
`25
`25
`25
`21
`Mean
`S.D.
`
`Mean
`S.D.
`
`1.81
`0.49
`0.44
`0.34
`0.45
`0.54
`0.67
`0.57
`0.52
`0.37
`0.62
`0.43
`
`0.71
`1.06
`0.66
`1.80
`1.61
`1.36
`1.20
`0.47
`
`0.84
`0.52
`
`3.09
`1.77
`1.45
`1.49
`1.42
`1.63
`3.03
`I. 71
`1.55
`1.72
`1.89
`0.63
`
`2.27
`5.25
`1.98
`5.14
`3.52
`2.86
`3.50
`1.41
`
`2.50
`1.25
`
`4.90
`2.26
`1.89
`1.83
`1.87
`2.17
`3.70
`2.28
`2.07
`2.09
`2.58
`1.12
`
`2.98
`6.31
`2.64
`6.94
`5.13
`4.22
`4.70
`1.75
`
`3.34
`1.68
`
`0.37
`0.22
`0.23
`0.18
`0.24
`0.25
`0.22
`0.25
`0.25
`0.18
`0.24
`0.05
`
`0.24
`0.17
`0.25
`0.26
`0.31
`0.32
`0.26
`0.05
`
`0.25
`0.05
`
`a) M, male; F, female. b) Conjugated PAA value is obtained by subtracting the free value from the
`total value.
`
`TABLE II. Plasma Concentrations of PAA (nmol/ml) in Control Rats
`
`Free
`
`Conjugate"'
`
`Total
`
`Free(total
`(mol/mol)
`
`I
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`Mean
`S.D.
`
`0.23
`0.51
`
`. 0.32
`
`0.33
`0.47
`0.31
`0.48
`0.32
`0.31
`0.19
`
`0.35
`0.11
`
`2.13
`3.54
`2.83
`2.00
`4.06
`1.81
`3.12
`2.56
`2.46
`1.31
`
`2.58
`0.83
`
`2.36
`4.05
`3.15
`2.33
`4.53
`2.12
`3.60
`2.88
`2.77
`1.50
`
`2.93
`0.33
`
`0.10
`0.13
`0.10
`0.14
`0.10
`0.15
`0.13
`0.11
`0.12
`0.13
`
`0.12
`0.02
`
`a) Conjugated PAA value is obtained by subtracting the free value from the total value.
`
`workers. 1
`4
`6
`•2
`> The data indicated that plasma PAA concentration in females is significantly
`•
`-
`higher than that in males {p<O.Ol). The same observation was also reported by Davis et af.Z>
`PAA Concentration in Rat Plasma
`It is known that rats and mice, forced to swim in water, show a characteristic posture.
`
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`

`

`No.9
`
`3745
`
`TABLE III. Plasma Concentration of Free and Total PAA (nmoljml)
`in "Behavioral Despair" Rats
`
`Free
`
`Conjugated">
`
`Total
`
`Free/total
`(mol/mol)
`
`I
`2
`3
`4
`5
`6
`7
`8
`9
`10
`
`Mean
`S.D.
`
`0.28
`0.18
`0.29
`0.43
`0.33
`0.38
`0.33
`0.36
`0.26
`0.36
`
`0.32
`O.o7
`
`1.73
`2.02
`2.13
`2.06
`1.76
`4.12
`3.31
`3.41
`1.77
`1.99
`
`2.43
`0.85
`
`2.01
`2.20
`2.42
`2.49
`2.09
`4.50
`3.64
`3.77
`2.03
`2.35
`
`2.75
`0.88
`
`0.14
`0.08
`0.12
`0.17
`0.16
`0.08
`0.09
`0.10
`0.13
`0.15
`
`0.12
`0.03
`
`a) Conjugated PAA value is obtained by subtracting the free value from the total value.
`
`Porsolt et a!. proposed this "behavioral despair" as an animal model of depression. 3 ) The
`concentrations of free and total PAA in plasma from control and "behavioral despair" rats
`determined by this method are shown in Table II. The levels ofPAA in rat plasma were first
`determined by the present HPLC method. As shown in this Table, the mean values of free and
`total P AA in "behavioral despair" rat plasma were only slightly lower than those in control
`rat plasma, and a t test showed that the values were not significantly lower than those of the
`controls.
`This study provides the first ftuorimetric HPLC method for the assay of PAA in human
`and rat sera. The present method is highly sensitive; the sensitivity permits the assay of free
`and total PAA using a minute amount of plasma (50,ul). Namely, the method allows the
`determination of PAA in plasma of an experimental small animal. Although it remains to be
`seen whether the measurement of P AA provides significant information on brain function,
`this method should allow us to address the problem. The method is rapid and simple to
`perform, and could therefore be applied for routine use for the diagnosis and monitoring of
`depressive illness.
`
`References
`
`I) F. Gusovsky, J. Fawcet, J. I. Javaid, H. Jeffriess and H. Sabelli, Anal. Biachem., 145, 101 (1985).
`2) B. A. Davis, D. A. Durden and A. A. Boulton, J. Chramatagr., 230, 219 (1982).
`3) R. D. Porsolt, G. Anton, N. Blavet and M. Jalfre, Eur. J. Pharmacal., 47, 379 (1978).
`4) L. E. Fellows, G. S. King, B. R. Pettit, B. L. Goodwin, C. R. J. Ruthven and M. Sandler, Biamed. Mass
`Spectram., 5, 508 (1978).
`5) A. A. Boulton, B. A. Davis, P. H. Yu, J. S. Wosmith and D. Addington, Psychiatry Res., 8, 19 (1983).
`6) M. Sandler, C. R. F. Ruthven, B. L. Goodwin, A. Lees and G. M. Stein, J. Neural. Neurasurg. Psychiatry, 45,
`366 (1982).
`7) B. A. Davis, P. H. Yu, K. Carlson, K. O'Sullivan and A. A. Boulton, Psychiatry Res., 6, 97 (1982).
`8) M. Yamaguchi, R. Matsunaga, K. Fukuda and M. Nakamura, J. Chramatagr., 414, 275 (1987).
`9) M. Yamaguchi, S. Hara, R. Matsunaga, M. Nakamura andY. Ohkura, J. Chromatogr., 346, 227 (1985).
`
`Par Pharmaceutical, Inc. Ex. 1025
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