Journal of Neuroscience Methods, 34 (1990) 17-22
17
Elsevier NSM 01102
Amino acid measurement by high-performance liquid chromatography using electrochemical detection * R o y A. Sherwood Department of Clinical Biochemistry, King's College Hospital, Denmark Hill, London (U.K.) (Received 1 December 1989) (Accepted 1 March 1990)
Key words: Arrfino acids; High performance liquid chromatography; Electrochemical detection; Phenylisothiocyanate The measurement of amino acids by high-performance liquid chromatography has become more common in recent years. Separation can be by gradient elution of the native amino acids followed by reaction with ninhydrin or by pre-column derivatisation with ultraviolet or fluorescent detection. The use of electrochemical detection for the measurement of amino acid derivatives formed with either o-phthalaldehyde or phenylisothiocyanate is discussed. A method for the assay of amino acids in human blood serum and urine samples using phenylisothiocyanate as the derivatising agent and electrochemical detection is described and compared to previously reported methods based on o-phthalaldehyde derivatisation.
Amino acid chromatography Amino acid analysis is an important procedure used by biochemists employed in a diverse range of activities including the neurosciences, the pharmaceutical industry and clinical chemistry. Traditionally thin-layer chromatography (TLC) and ion-exchange chromatography have been used for the detection and quantitation of amino acids in a wide range of samples. TLC has been primarily used as a qualitative or semi-quantitative technique while ion exchange chromatography forms the basis for most commercial amino acid analysers. The advances in technology in the field
* Presented at: Electrochemieal Detection, HPLC and In Vivo Monitoring in the Biosciences, Nottingham University, 19-21 September, 1989. Correspondence: R.A. Sherwood, Department of Clinical Biochemistry, Kings College Hospital, Denmark Hill, London SE5 91~, U.K.
of high-performance liquid chromatography (HPLC) have led to an increasing interest in HPLC as a means of measuring amino acids. The majority of HPLC methods so far described for quantitation of the forty or so amino acids of interest to the researcher in the biomedical field need gradient elution to resolve such a large number of components adequately. Although separation of the native amino acids is achievable by gradient ion-exchange chromatography (usually visualised by post-column reaction with ninhydrin) most workers have formed derivatives prior to a HPLC separation. A number of methods for derivatisation of amino acids have been reported to be suitable for subsequent separation by reversed phase HPLC. The most commonly used are shown below: (1) Dansyl chloride (Rutledge and Rudy, 1987) (2) Dabsyl chloride (Lin and Wang, 1980) (3) 9-Fluorenylmethyl chloroformate (FMOC) (Einarsson et al., 1982)
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
18 (4) o-Phthalaldehyde (OPA) (Lindroth and Mopper, 1979) (5) Phenylisothiocyanate (PITC) (Bidlingmeyer et al., 1984) Most of the above-mentioned derivatives have some disadvantages either generally or when used with particular sample media. These disadvantages relate to the properties of the derivatives, the complexity of the derivatisation process or to the means of detection. The first four derivatisation methods listed above produce derivatives of the amino acids that can be detected by their fluorescence which although a sensitive method of detection, can introduce problems. Both dansyl and dabsyl chloride derivatisation give poor yields unless added in considerable excess (Lin and Wang, 1980; Rutledge and Rudy, 1987). This in turn necessitates an extraction step to remove the dansyl sulphonic acid compounds which are by-products of the reaction and which can produce considerable interference if allowed to remain during the chromatography step. A similar problem occurs with the use of 9-fluorenylmethyl chloroformate as it forms fluorenylmethyl alcohol which must be removed prior to analysis (Einarsson et al., 1982). The derivatisation reagent which has been most often used is OPA which is itself non-fluorescent and does not give fluorescent by-products (Lindroth and Mopper, 1979; Turnell and Cooper, 1982). Most primary amino acids form substituted indole products which are fluorescent, but major disadvantages of the method include instability of the iso-indoles and the non-reactivity of OPA with the secondary amino acids. As the measurement of the imino acids proline and hydroxyproline is often required, particularly in the clinical chemistry field this is a significant disadvantage. Proline can be measured if hypoehlorite is added as a post-column reagent but this has a side effect of lowering the sensitivity of the method to most other amino acids. Transient addition of the hypochlorite partially overcomes this but adds to the complexity of the overall system. A derivatisation reagent which overcomes many of these problems is phenylisothiocyanate (PITC), often referred to as the Edman reagent (Bidlingmeyer et al., 1984). This reacts with both
primary and secondary amino acids to form phenylthiocarbamyl (PTC) derivatives that can be detected by their ultraviolet (UV) absorption at 254 nm (Ebert, 1986; Janssen et al., 1986; O'Hare et al., 1987). Whilst this reagent overcomes the problems of non-reactivity with secondary amino acids encountered using OPA, the use of 254 nm as the wavelength for detection poses its own problems, particularly with biological samples. Unreacted PITC absorbs at this wavelength as do many compounds found in blood or urine samples and removal of the interfering substances must be carried out prior to analysis. UV detection is also usually less sensitive than either fluorescence or electrochemical detection. Despite these problems both OPA with fluorescent detection and PITC with UV detection have been used for the measurement of amino acids in a variety of applications.
Electrochemical detection of amino acid derivatives
Joseph and Davies (1983) were the first to take advantage of the electroactivity of the isoindole nucleus of OPA to produce a HPLC method for the assay of amino acids with electrochemical detection. Other workers have since shown an interest in this technique (Zielke, 1985). The formation of OPA derivatives of amino acids requires the presence of a thiol, typically 2-mercaptoethanol (Joseph and Davies, 1983). The stability of the derivatives varies significantly with both the amine structure and the size of the thiol group due to steric hindrance of OPA degradation. A number of different thiols have been tried but in some cases although the stability of the derivatives was improved, the intensity of fluorescence emitted also fell to 10-15% of that emitted by the OPA/2-mercaptoethanol combination (Allison et al., 1983). The redox behaviour is, however, relatively insensitive to changes in the thiol used. Recently sodium sulphite has been recommended as the thiol of choice for OPA derivatisation if only electrochemical detection is to be used (Jacobs, 1987). The OPA-sulphite derivatives formed have superior stability to those described previously with no apparent loss of activity over a
19
30-min period, although the derivatives are subject to hydrolysis at low pH (Jacobs, 1987). Whilst electrochemical detection of OPA derivatives has permitted the use of reagents" which confer greater stability, OPA still has the disadvantage of not reacting with secondary amino acids. Granberg (1984) reported that PITC derivatives of amino acids were electrochemically active. In a paper concerning the measurement of the amino acid composition of an insulin hydrolysate he described both UV and electrochemical detection of the PTC derivatives. Recently the application of electrochemical detection of PTC-amino acids to the detection and quantitation of amino acids in body fluids has been reported (Sherwood et al., 1989a).
TABLE I THE GRADIENT PROFILE USED FOR SEPARATION OF PITC DERIVATIVES O F A M I N O A C I D S Buffer A is 0.05 M sodium acetate, p H 6.40; Buffer B is 60% 0.05 M sodium acetate, p H 6.40, 40% acetonitrile. Time
Buffer A
Buffer B
Flow
(rain)
(%)
(%)
(n~/min)
0 20 65 67.5 70 75 80
100 87 45 0 0 100 100
0 13 55 100 100 0 0
1.0 1.0 1.0 2.0 2.0 2.0 2.0
derivatisation. The PTC derivatives were formed
by reacting the dry residue of deproteinised serum Methodology Blood and urine samples (100 #1) were deproteinized with methanol (400 #1), centrifuged and the organic phase evaporated to dryness prior to
or urine or untreated standard (10/~1) with PITC (10 /d of PITC/methanol, 1:8) and a coupling solvent such as triethylamine (200 /~1 of triethylamine, 1:10). This was mixed and allowed to react for 5 min before evaporation using a rotary evaporator. The dried residue was reconstituted
Buffer B
ii
100
m z
m c~
s0
i
W
w z tam
c~
tu
u~
Q
~
g
e4 Z
i 1
J
Time (rain) Fig. 1. H P L C - E C separation by gradient elution of the 17 amino acid standard following PITC derivatisation. The change in the buffer composition is shown as the percentage of Buffer B. IS = norleucine. For analytical conditions see Methods. Sensitivity 3/~A full scale.
20 with mobile phase (500 /xl of buffer A, 0.05 M sodium acetate, p H 6.4). T o remove any remaining P I T C or interfering substances dichloromethane (200 /H) was added, mixed for 1 min using a vortex mixer and centrifuged at 3000 r p m for 1 - 3 rain. The aqueous phase was then ready for injection into a gradient H P L C system. The H P L C system used was a SP8700 X R tertiary gradient p u m p (Spectraphysics, St. A1bans, U.K.), O D S Hypersil cartridge column (250 × 4.6 ram, 5 p m , from Jones C h r o m a t o g r a p h y , Hengoed, U.K.) and an L C A 15 electrochemical detector ( E D T Research, L o n d o n , U.K.). The output was monitored using a C H R O M J E T computing integrator (Spectraphysics, St. Albans, U.K.). A R h e o d y n e injection valve with a 20-ttl sample loop was used to inject the sample. The gradient was formed between buffer A (0.05 M sodium acetate, p H 6.40) and buffer B (0.05 M sodium acetate, p H 6.40, containing 40% acetonitrile) as shown in Table I. The complete analytical cycle took 80 rain. Electrochemical detection took place with an operating potential of + 1.1 V (Glassy c a r b o n electrode vs. A g / A g C 1 reference electrode) and a sensitivity of 3 #A. A n internal standard (10 /~1 of norleucine) was added when quantitation was required. Results and discussion A c h r o m a t o g r a m of a standard mixture of 17 amino acids is shown in Fig. 1. The mean retention times and retention times relative to norleucine for the 40 most c o m m o n l y measured amino acids are shown in Table II. The resolution obtained is satisfactory and is sufficient to cope with virtually all the clinical situations where qualitative or quantitative amino acid assay is needed. It is often stated that gradient elution produces difficulties when using H P L C with electrochemical detection. The m e t h o d described here with P I T C derivatisation and that described b y Joseph and Davies (1983) b o t h successfully use gradient elution and electrochemical detection for the measurement of amino acid derivatives. Providing the organic phase concentration is not increased too rapidly the base lines obtained are suitable for normal use as shown in Fig. 1.
TABLE II THE RETENTION TIMES AND RELATIVE RETENTION TIMES FOR THE 40 MOST COMMONLY MEASURED AMINO ACIDS The times are a compilation of the mean times obtained for 10 repeat analyses of a mixed standard. Amino acid
1. Phosphoserine 2. Aspartic acid 3. Glutamic acid 4. -/-Amino adipic acid 5. Hydroxyproline 6. Phosphoethanolamine 7. Serine 8. Glycine 9. Asparagine 10. Sarcosine 11. fl-Alanine 12. Taurine 13. -/-Aminobutyric acid 14. Threonine 15. Citrulline 16. Alanine 17. fl-Arainoisobutyric acid 18. Proline 19. Histidine 20. Carnosine 21. Arginine 22. Anserine 23.1 -Methylhistidine 24. 3-Methylhistidine 25. a-Aminobutyric acid 26. Tyrosine 27. Valine 28. Ethanolamine 29. Methionine 30. Cystathionine 31. Cystine 32. Isoleucine 33. Leucine 34. Nofleucine 35. Hydroxylysine 1 36. Hydroxylysine 2 37. Phenylalanine 38. Ornithine 39. Tryptophan 40. Lysine
Retention times (mean RT) 3.82 5.44 6.83 10.35 12.26 12.45 14.52 15.52 15.54 16.91 17.42 18.98 20.48 21.11 20.63 21.75 22.27 23.48 23.91 25.71 28.22 29.06 28.39 28.39 28.60 35.02 35.76 37.65 38.10 38.19 40.88 42.80 43.47 45.07 47.78 48.46 48.20 49.67 50.16 52.79
Relative retention times (mean RT) 0.084 0.120 0.150 0.229 0.271 0.273 0.321 0.342 0.343 0.373 0.384 0.419 0.452 0.465 0.462 0.481 0.492 0.525 0.528 0.568 0.623 0.628 0.625 0.627 0.632 0.774 0.794 0.822 0.841 0.846 0.906 0.950 0.965 1.055 1.070 1.069 1.097 1.107 1.169
The sensitivity of the P T C - a m i n o acid derivatives is considerably higher than that of O P A derivatives using electrochemical detection. Joseph
21
and Davies (1983) reported that OPA derivatives of plasma amino acids could be measured electrochemically with a full scale deflection of 10-50 nat. These workers, however, used operating potentials of +0.4 to +0.6 V Ag/AgC1 rather than the + 1.1 V necessary for PITC derivations. This was to achieve satisfactory specificity with their derivatisation procedures. A comparable amino acid standard (100/~mol/1) derivatised with PITC gave a full scale deflection of 1-3/LA using the method described above. The PITC derivatisation method described here has been used to assay amino acids in samples of blood and urine from patients with various inherited diseases of amino acid metabolism, utilising + 1.1 V as the working potential (Sherwood et al., 1989b). At a sensitivity setting of 3 #A no significant interference from endogenous electroactive compounds has been seen although the high applied voltage might have been expected to result in interference from compounds such as the biogenic amines. Closer examination reveals that even if the chromatographic conditions resulted in the retention of such compounds their activity is 1001000 times less than that seen for PTC-amino acid derivatives. Drugs, such as the penicillins, which have been shown to interfere with methods based on the ninhydrin reaction do not cause a problem with this system. The stability of the PTC-derivatives is considerably greater than that reported for any of the OPA methods. Samples prepared with PITC were stable at 4 °C for up to a week and appear to be stable indefinitely at - 2 0 ° C , unlike OPA derivatives where stability is measured in minutes or hours (Jacobs, 1987). Electrochemical detection has now been shown to be a viable alternative to fluorescence for both qualitative and quantitative assay of amino acids in biological fluids. The choice of OPA or PITC derivatisation is dependent on the application for which the method is developed, although if proline or hydroxyproline measurements are required then PITC must be the method of choice. The additional sensitivity offered by PITC is another significant advantage. Gradient elution has now been shown to be suitable for use with electrochemical detection allowing methods to be devel-
oped for specific amino acids with considerably shorter run times. Amino acids must therefore be added to the growing list of compounds that can be measured routinely by the combination of HPLC and electrochemical detection.
References Allison, L.A., Mayer, G.S. and Shoup, R.E. (1984) o-Phthalaldehyde derivatives of amines for high-speed liquid chromatography/electrochemistry, Anal. Chem., 56 1089-1096. Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1984) Rapid analysis of amino acids using pre-column derivatization, J. Chromatogr., 336: 93-104. Ebert, R.F. (1986) Amino acid analysis by HPLC: optimized conditions for chromatography of phenylthiocarbamyl derivatives, Anal. Biochem., 154: 431-435. Einarsson, S., Josefsson, B. and Lagerkvist, S. (1983) Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase high-performance fiquid chromatography, J. Chromatogr., 282: 609-618. Granberg, R.R. (1984) High resolution analysis of PITC-derivatized amino acids with UV and electrochemical detection, LC Mag., 2: 776-781. Jacobs, W.A. (1987) o-Phthalaldehyde-sulfite derivatization of primary amines for liquid chromatography-electrochemistry, J. Chromatogr., 392: 435-441. Janssen, P.S.L., Van Nispen, J.W., Melgers, P.A.T.A., Van der Bogaart, H.W.M., Van Aalst, G.W.M. and Goverde, B.C. (1986) HPLC analysis of PTC amino acids, II. Application in the analysis of polypeptides, Chromatographia, 22: 351357. Joseph, M.H. and Davies, P. (1983) Electrochemical activity of o-phthalaldehyde-mercaptoethanol derivatives of amino acids, J. Chromatogr. Biomed. Appl., 277: 125-136. Lin, J.-K. and Wang, C.-H. (1980) Determination of urinary amino acids by liquid chromatography with 'Dabsyl chloride', Clin. Chem., 26: 579-583. Lindroth, P. and Mopper, K. (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by pre-colunm fluorescence derivatisation with o-phthalaldehyde, Anal. Chem., 51: 1667-1674. O'Hare, M.M.T., Tortora, O., Gether, U., Nielsen, H.V. and Schwartz, T.W. (1987) High-performance liquid chromatography of phenylthiocarbamyl derivatives of amino acids and side-chain derivatized amino acids, J. Chromatogr., 389: 379-388. Rutledge, J.C. and Rudy, J. (1987) HPLC qualitative amino acid analysis in the clinical laboratories, Am. J. Clin. Pathol., 87: 614-618. Sherwood, R.A., Richards, D.R. and Titheradge, A.C. (1989a) Analysis of amino acids in human plasma and urine by HPLC with electrochemical detection. Neurosci. Methods 29: 270.
22 Sherwood, R.A., Richards, D.R. and Titheradge, A.C. (1989b) Measurement of plasma and urine amino acids by high performance liquid chromatography with electrochemical detection using PITC derivatisation, J. Chromatogr. Biomed. Appl., In press. TurneU, D.C. and Cooper, J.D.H. (1982) Rapid assay for amino acids in serum or urine by pre-column derivatization
and reversed-phase liquid chromatography, Clin. Chem., 28: 527-531. Zielke, H.R. (1985) Determination of amino acids in the brain by high performance liquid chromatography with isocratic elution and electrochemical detection, J. Chromatogr., 347: 320-324.
17
Elsevier NSM 01102
Amino acid measurement by high-performance liquid chromatography using electrochemical detection * R o y A. Sherwood Department of Clinical Biochemistry, King's College Hospital, Denmark Hill, London (U.K.) (Received 1 December 1989) (Accepted 1 March 1990)
Key words: Arrfino acids; High performance liquid chromatography; Electrochemical detection; Phenylisothiocyanate The measurement of amino acids by high-performance liquid chromatography has become more common in recent years. Separation can be by gradient elution of the native amino acids followed by reaction with ninhydrin or by pre-column derivatisation with ultraviolet or fluorescent detection. The use of electrochemical detection for the measurement of amino acid derivatives formed with either o-phthalaldehyde or phenylisothiocyanate is discussed. A method for the assay of amino acids in human blood serum and urine samples using phenylisothiocyanate as the derivatising agent and electrochemical detection is described and compared to previously reported methods based on o-phthalaldehyde derivatisation.
Amino acid chromatography Amino acid analysis is an important procedure used by biochemists employed in a diverse range of activities including the neurosciences, the pharmaceutical industry and clinical chemistry. Traditionally thin-layer chromatography (TLC) and ion-exchange chromatography have been used for the detection and quantitation of amino acids in a wide range of samples. TLC has been primarily used as a qualitative or semi-quantitative technique while ion exchange chromatography forms the basis for most commercial amino acid analysers. The advances in technology in the field
* Presented at: Electrochemieal Detection, HPLC and In Vivo Monitoring in the Biosciences, Nottingham University, 19-21 September, 1989. Correspondence: R.A. Sherwood, Department of Clinical Biochemistry, Kings College Hospital, Denmark Hill, London SE5 91~, U.K.
of high-performance liquid chromatography (HPLC) have led to an increasing interest in HPLC as a means of measuring amino acids. The majority of HPLC methods so far described for quantitation of the forty or so amino acids of interest to the researcher in the biomedical field need gradient elution to resolve such a large number of components adequately. Although separation of the native amino acids is achievable by gradient ion-exchange chromatography (usually visualised by post-column reaction with ninhydrin) most workers have formed derivatives prior to a HPLC separation. A number of methods for derivatisation of amino acids have been reported to be suitable for subsequent separation by reversed phase HPLC. The most commonly used are shown below: (1) Dansyl chloride (Rutledge and Rudy, 1987) (2) Dabsyl chloride (Lin and Wang, 1980) (3) 9-Fluorenylmethyl chloroformate (FMOC) (Einarsson et al., 1982)
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
18 (4) o-Phthalaldehyde (OPA) (Lindroth and Mopper, 1979) (5) Phenylisothiocyanate (PITC) (Bidlingmeyer et al., 1984) Most of the above-mentioned derivatives have some disadvantages either generally or when used with particular sample media. These disadvantages relate to the properties of the derivatives, the complexity of the derivatisation process or to the means of detection. The first four derivatisation methods listed above produce derivatives of the amino acids that can be detected by their fluorescence which although a sensitive method of detection, can introduce problems. Both dansyl and dabsyl chloride derivatisation give poor yields unless added in considerable excess (Lin and Wang, 1980; Rutledge and Rudy, 1987). This in turn necessitates an extraction step to remove the dansyl sulphonic acid compounds which are by-products of the reaction and which can produce considerable interference if allowed to remain during the chromatography step. A similar problem occurs with the use of 9-fluorenylmethyl chloroformate as it forms fluorenylmethyl alcohol which must be removed prior to analysis (Einarsson et al., 1982). The derivatisation reagent which has been most often used is OPA which is itself non-fluorescent and does not give fluorescent by-products (Lindroth and Mopper, 1979; Turnell and Cooper, 1982). Most primary amino acids form substituted indole products which are fluorescent, but major disadvantages of the method include instability of the iso-indoles and the non-reactivity of OPA with the secondary amino acids. As the measurement of the imino acids proline and hydroxyproline is often required, particularly in the clinical chemistry field this is a significant disadvantage. Proline can be measured if hypoehlorite is added as a post-column reagent but this has a side effect of lowering the sensitivity of the method to most other amino acids. Transient addition of the hypochlorite partially overcomes this but adds to the complexity of the overall system. A derivatisation reagent which overcomes many of these problems is phenylisothiocyanate (PITC), often referred to as the Edman reagent (Bidlingmeyer et al., 1984). This reacts with both
primary and secondary amino acids to form phenylthiocarbamyl (PTC) derivatives that can be detected by their ultraviolet (UV) absorption at 254 nm (Ebert, 1986; Janssen et al., 1986; O'Hare et al., 1987). Whilst this reagent overcomes the problems of non-reactivity with secondary amino acids encountered using OPA, the use of 254 nm as the wavelength for detection poses its own problems, particularly with biological samples. Unreacted PITC absorbs at this wavelength as do many compounds found in blood or urine samples and removal of the interfering substances must be carried out prior to analysis. UV detection is also usually less sensitive than either fluorescence or electrochemical detection. Despite these problems both OPA with fluorescent detection and PITC with UV detection have been used for the measurement of amino acids in a variety of applications.
Electrochemical detection of amino acid derivatives
Joseph and Davies (1983) were the first to take advantage of the electroactivity of the isoindole nucleus of OPA to produce a HPLC method for the assay of amino acids with electrochemical detection. Other workers have since shown an interest in this technique (Zielke, 1985). The formation of OPA derivatives of amino acids requires the presence of a thiol, typically 2-mercaptoethanol (Joseph and Davies, 1983). The stability of the derivatives varies significantly with both the amine structure and the size of the thiol group due to steric hindrance of OPA degradation. A number of different thiols have been tried but in some cases although the stability of the derivatives was improved, the intensity of fluorescence emitted also fell to 10-15% of that emitted by the OPA/2-mercaptoethanol combination (Allison et al., 1983). The redox behaviour is, however, relatively insensitive to changes in the thiol used. Recently sodium sulphite has been recommended as the thiol of choice for OPA derivatisation if only electrochemical detection is to be used (Jacobs, 1987). The OPA-sulphite derivatives formed have superior stability to those described previously with no apparent loss of activity over a
19
30-min period, although the derivatives are subject to hydrolysis at low pH (Jacobs, 1987). Whilst electrochemical detection of OPA derivatives has permitted the use of reagents" which confer greater stability, OPA still has the disadvantage of not reacting with secondary amino acids. Granberg (1984) reported that PITC derivatives of amino acids were electrochemically active. In a paper concerning the measurement of the amino acid composition of an insulin hydrolysate he described both UV and electrochemical detection of the PTC derivatives. Recently the application of electrochemical detection of PTC-amino acids to the detection and quantitation of amino acids in body fluids has been reported (Sherwood et al., 1989a).
TABLE I THE GRADIENT PROFILE USED FOR SEPARATION OF PITC DERIVATIVES O F A M I N O A C I D S Buffer A is 0.05 M sodium acetate, p H 6.40; Buffer B is 60% 0.05 M sodium acetate, p H 6.40, 40% acetonitrile. Time
Buffer A
Buffer B
Flow
(rain)
(%)
(%)
(n~/min)
0 20 65 67.5 70 75 80
100 87 45 0 0 100 100
0 13 55 100 100 0 0
1.0 1.0 1.0 2.0 2.0 2.0 2.0
derivatisation. The PTC derivatives were formed
by reacting the dry residue of deproteinised serum Methodology Blood and urine samples (100 #1) were deproteinized with methanol (400 #1), centrifuged and the organic phase evaporated to dryness prior to
or urine or untreated standard (10/~1) with PITC (10 /d of PITC/methanol, 1:8) and a coupling solvent such as triethylamine (200 /~1 of triethylamine, 1:10). This was mixed and allowed to react for 5 min before evaporation using a rotary evaporator. The dried residue was reconstituted
Buffer B
ii
100
m z
m c~
s0
i
W
w z tam
c~
tu
u~
Q
~
g
e4 Z
i 1
J
Time (rain) Fig. 1. H P L C - E C separation by gradient elution of the 17 amino acid standard following PITC derivatisation. The change in the buffer composition is shown as the percentage of Buffer B. IS = norleucine. For analytical conditions see Methods. Sensitivity 3/~A full scale.
20 with mobile phase (500 /xl of buffer A, 0.05 M sodium acetate, p H 6.4). T o remove any remaining P I T C or interfering substances dichloromethane (200 /H) was added, mixed for 1 min using a vortex mixer and centrifuged at 3000 r p m for 1 - 3 rain. The aqueous phase was then ready for injection into a gradient H P L C system. The H P L C system used was a SP8700 X R tertiary gradient p u m p (Spectraphysics, St. A1bans, U.K.), O D S Hypersil cartridge column (250 × 4.6 ram, 5 p m , from Jones C h r o m a t o g r a p h y , Hengoed, U.K.) and an L C A 15 electrochemical detector ( E D T Research, L o n d o n , U.K.). The output was monitored using a C H R O M J E T computing integrator (Spectraphysics, St. Albans, U.K.). A R h e o d y n e injection valve with a 20-ttl sample loop was used to inject the sample. The gradient was formed between buffer A (0.05 M sodium acetate, p H 6.40) and buffer B (0.05 M sodium acetate, p H 6.40, containing 40% acetonitrile) as shown in Table I. The complete analytical cycle took 80 rain. Electrochemical detection took place with an operating potential of + 1.1 V (Glassy c a r b o n electrode vs. A g / A g C 1 reference electrode) and a sensitivity of 3 #A. A n internal standard (10 /~1 of norleucine) was added when quantitation was required. Results and discussion A c h r o m a t o g r a m of a standard mixture of 17 amino acids is shown in Fig. 1. The mean retention times and retention times relative to norleucine for the 40 most c o m m o n l y measured amino acids are shown in Table II. The resolution obtained is satisfactory and is sufficient to cope with virtually all the clinical situations where qualitative or quantitative amino acid assay is needed. It is often stated that gradient elution produces difficulties when using H P L C with electrochemical detection. The m e t h o d described here with P I T C derivatisation and that described b y Joseph and Davies (1983) b o t h successfully use gradient elution and electrochemical detection for the measurement of amino acid derivatives. Providing the organic phase concentration is not increased too rapidly the base lines obtained are suitable for normal use as shown in Fig. 1.
TABLE II THE RETENTION TIMES AND RELATIVE RETENTION TIMES FOR THE 40 MOST COMMONLY MEASURED AMINO ACIDS The times are a compilation of the mean times obtained for 10 repeat analyses of a mixed standard. Amino acid
1. Phosphoserine 2. Aspartic acid 3. Glutamic acid 4. -/-Amino adipic acid 5. Hydroxyproline 6. Phosphoethanolamine 7. Serine 8. Glycine 9. Asparagine 10. Sarcosine 11. fl-Alanine 12. Taurine 13. -/-Aminobutyric acid 14. Threonine 15. Citrulline 16. Alanine 17. fl-Arainoisobutyric acid 18. Proline 19. Histidine 20. Carnosine 21. Arginine 22. Anserine 23.1 -Methylhistidine 24. 3-Methylhistidine 25. a-Aminobutyric acid 26. Tyrosine 27. Valine 28. Ethanolamine 29. Methionine 30. Cystathionine 31. Cystine 32. Isoleucine 33. Leucine 34. Nofleucine 35. Hydroxylysine 1 36. Hydroxylysine 2 37. Phenylalanine 38. Ornithine 39. Tryptophan 40. Lysine
Retention times (mean RT) 3.82 5.44 6.83 10.35 12.26 12.45 14.52 15.52 15.54 16.91 17.42 18.98 20.48 21.11 20.63 21.75 22.27 23.48 23.91 25.71 28.22 29.06 28.39 28.39 28.60 35.02 35.76 37.65 38.10 38.19 40.88 42.80 43.47 45.07 47.78 48.46 48.20 49.67 50.16 52.79
Relative retention times (mean RT) 0.084 0.120 0.150 0.229 0.271 0.273 0.321 0.342 0.343 0.373 0.384 0.419 0.452 0.465 0.462 0.481 0.492 0.525 0.528 0.568 0.623 0.628 0.625 0.627 0.632 0.774 0.794 0.822 0.841 0.846 0.906 0.950 0.965 1.055 1.070 1.069 1.097 1.107 1.169
The sensitivity of the P T C - a m i n o acid derivatives is considerably higher than that of O P A derivatives using electrochemical detection. Joseph
21
and Davies (1983) reported that OPA derivatives of plasma amino acids could be measured electrochemically with a full scale deflection of 10-50 nat. These workers, however, used operating potentials of +0.4 to +0.6 V Ag/AgC1 rather than the + 1.1 V necessary for PITC derivations. This was to achieve satisfactory specificity with their derivatisation procedures. A comparable amino acid standard (100/~mol/1) derivatised with PITC gave a full scale deflection of 1-3/LA using the method described above. The PITC derivatisation method described here has been used to assay amino acids in samples of blood and urine from patients with various inherited diseases of amino acid metabolism, utilising + 1.1 V as the working potential (Sherwood et al., 1989b). At a sensitivity setting of 3 #A no significant interference from endogenous electroactive compounds has been seen although the high applied voltage might have been expected to result in interference from compounds such as the biogenic amines. Closer examination reveals that even if the chromatographic conditions resulted in the retention of such compounds their activity is 1001000 times less than that seen for PTC-amino acid derivatives. Drugs, such as the penicillins, which have been shown to interfere with methods based on the ninhydrin reaction do not cause a problem with this system. The stability of the PTC-derivatives is considerably greater than that reported for any of the OPA methods. Samples prepared with PITC were stable at 4 °C for up to a week and appear to be stable indefinitely at - 2 0 ° C , unlike OPA derivatives where stability is measured in minutes or hours (Jacobs, 1987). Electrochemical detection has now been shown to be a viable alternative to fluorescence for both qualitative and quantitative assay of amino acids in biological fluids. The choice of OPA or PITC derivatisation is dependent on the application for which the method is developed, although if proline or hydroxyproline measurements are required then PITC must be the method of choice. The additional sensitivity offered by PITC is another significant advantage. Gradient elution has now been shown to be suitable for use with electrochemical detection allowing methods to be devel-
oped for specific amino acids with considerably shorter run times. Amino acids must therefore be added to the growing list of compounds that can be measured routinely by the combination of HPLC and electrochemical detection.
References Allison, L.A., Mayer, G.S. and Shoup, R.E. (1984) o-Phthalaldehyde derivatives of amines for high-speed liquid chromatography/electrochemistry, Anal. Chem., 56 1089-1096. Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L. (1984) Rapid analysis of amino acids using pre-column derivatization, J. Chromatogr., 336: 93-104. Ebert, R.F. (1986) Amino acid analysis by HPLC: optimized conditions for chromatography of phenylthiocarbamyl derivatives, Anal. Biochem., 154: 431-435. Einarsson, S., Josefsson, B. and Lagerkvist, S. (1983) Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase high-performance fiquid chromatography, J. Chromatogr., 282: 609-618. Granberg, R.R. (1984) High resolution analysis of PITC-derivatized amino acids with UV and electrochemical detection, LC Mag., 2: 776-781. Jacobs, W.A. (1987) o-Phthalaldehyde-sulfite derivatization of primary amines for liquid chromatography-electrochemistry, J. Chromatogr., 392: 435-441. Janssen, P.S.L., Van Nispen, J.W., Melgers, P.A.T.A., Van der Bogaart, H.W.M., Van Aalst, G.W.M. and Goverde, B.C. (1986) HPLC analysis of PTC amino acids, II. Application in the analysis of polypeptides, Chromatographia, 22: 351357. Joseph, M.H. and Davies, P. (1983) Electrochemical activity of o-phthalaldehyde-mercaptoethanol derivatives of amino acids, J. Chromatogr. Biomed. Appl., 277: 125-136. Lin, J.-K. and Wang, C.-H. (1980) Determination of urinary amino acids by liquid chromatography with 'Dabsyl chloride', Clin. Chem., 26: 579-583. Lindroth, P. and Mopper, K. (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by pre-colunm fluorescence derivatisation with o-phthalaldehyde, Anal. Chem., 51: 1667-1674. O'Hare, M.M.T., Tortora, O., Gether, U., Nielsen, H.V. and Schwartz, T.W. (1987) High-performance liquid chromatography of phenylthiocarbamyl derivatives of amino acids and side-chain derivatized amino acids, J. Chromatogr., 389: 379-388. Rutledge, J.C. and Rudy, J. (1987) HPLC qualitative amino acid analysis in the clinical laboratories, Am. J. Clin. Pathol., 87: 614-618. Sherwood, R.A., Richards, D.R. and Titheradge, A.C. (1989a) Analysis of amino acids in human plasma and urine by HPLC with electrochemical detection. Neurosci. Methods 29: 270.
22 Sherwood, R.A., Richards, D.R. and Titheradge, A.C. (1989b) Measurement of plasma and urine amino acids by high performance liquid chromatography with electrochemical detection using PITC derivatisation, J. Chromatogr. Biomed. Appl., In press. TurneU, D.C. and Cooper, J.D.H. (1982) Rapid assay for amino acids in serum or urine by pre-column derivatization
and reversed-phase liquid chromatography, Clin. Chem., 28: 527-531. Zielke, H.R. (1985) Determination of amino acids in the brain by high performance liquid chromatography with isocratic elution and electrochemical detection, J. Chromatogr., 347: 320-324.
