- Email: [email protected]
[5] Quantitative high-performance liquid chromatographic analysis of branched-chain 2-keto acids in biological samples
20
ANALYTICAL
AND SYNTHETIC
METHODS
[5]
[5] Quantitative High-Performance Liquid Chromatographic Analysis of Branched-Chain 2-Keto Acids in Biological Samples By ANGELA L. GASKING, WILLIAM T. E. EDWARDS, ANTHONY HOBSON-FROHOCK,MARINOS ELIA, and GEOFFREYLIVESEY The concentrations o f the branched-chain 2-keto acids, 4-methyl-2-ketovaleric acid (ketoleucine), 3-methyl-2-ketovaleric acid (ketoisoleucine), and 3-methyl-2-ketobutyric acid (ketovaline), in m a m m a l i a n tissues are small, |-3 making quantification difficult. The major tissue pools o f keto acids in the rat are plasma (and whole blood) and muscle. 2,3 In h u m a n blood the keto acids predominate in p l a s m # and partly in association with albumin. 5 H u m a n urine normally contains amounts o f these keto acids which are too small to quantify with satisfactory accuracy. Analysis o f the branched-chain 2-keto acids is important in biochemistry and clinical chemistry with concentrations in blood or plasma being elevated by starvation and diabetes 4 and several disorders o f branched-chain amino acid metabolism. 6 Several analytical methods have been developed to quantify the branched-chain 2-keto acids using gas chromatography, ~,7 high-performance liquid chromatography, s- 10 or enzymatic determination. 2 The enzymatic m e t h o d is rapid and reliable and sufficiently sensitive for analysis o f the keto acids in muscle, plasma, and whole blood, but does not distinguish between the three individual acids. A highly sensitive high-performance liquid chromatographic method is described below involving a single derivatization procedure to the stable quinoxalinols which are detected by fluorescence. The method is based on that o f Hayashi and co-workers, 8,9 with a modification to exclude oxygen during derivatization. 1° See also P. L. Crowell, R. H. Miller, and A. E. Harper, this volume [7]. 2G. Liveseyand P. Lund, Biochem. J. 188, 705 (1980);see also G. Liveseyand P. Lund, this volume [ 1]. 3S. M. Hutson and A. E. Harper, Am. J. Clin. Nutr. 34,, 173 (!981). 4 M. Elia and G. Livesey,Clin. Sci. 64, 517 (1983). 5G. Liveseyand P. Lund~Biochem. J. 212, 655 (1983). K. Tanaka and L. E. Rosenberg, in "The Metabolic Basis of Inherited Diseases" (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredriekson,J. L. Goldstein, and M. S. Brown, eds.), p. 440. McGraw-Hill,New York, 1983. 7T. C. Cree, S. M. Hutson, and A. E. Harper, Anal Biochem. 92, 156 (1979). a T. Hayashi, T. Hironori, H. Todoriki, and H. Naruse, AnaL Biochem. 122, 173 (1982). 9 T. Hayashi, H. Tsuehiya, and H. Naruse, J. Chromatogr. 273, 245 (1983). l0 G. Liveseyand W. T. E. Edwards, J. Chromatogr. 337, 98 (1985).
METHODS IN ENZYMOLOGY, VOL. 166
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
QUANTITATIVE H P L C OF KETO ACIDS
21
Principles of the Method The proteins of blood and plasma are precipitated with acid and the solution is purified by retention of the keto acids on a hydrazide gel. The hydrazones are converted anaerobically to the quinoxalinols with ophenylenediamine which are then separated and quantified by HPLC using a reversed-phase column and a fluorescence detector. Gel - CONH- NH 2 + R. CO" CO2H--*Gel- CONHN:CR- CO2H OH GeI-CONHN:CR.CO2H + ~
~
~ GeI-CONH.NH 2 +
~ N H 2
N
R
4-Methyl-2-ketovaleric acid [R = --CH2CH2CH(CH3) 2 3-Methyl-2-ketovaleric acid [R = --CH2CH(CH3)CH2CH3] 3-Methyl-2-ketobutyric acid [R = --CH2CH(CH3)2]
Preparation of the Hydrazide Gel Reagents
BioGel P-60, 100-200 mesh (Bio-Rad Ltd) Hydrazine hydrate, 98% Sodium chloride, 0.1 M Borate buffer, 0.1 M H2BO3 containing 0.2 M NaC1, 0.02 M disodium ethylenediaminetetraacetic acid (Na2 EDTA), 5 #M pentachlorophenol (final concentrations) to pH 7.3 with NaOH 'The preparative procedure is based on the method of Hayashi et al. 9 Dry gel (15 g) is added to water (200 ml) in a siliconized flask and left overnight. The gel suspension is heated to 50 ° and mixed with 80 ml of the hydrazine hydrate also at 50 °. The mixture is stirred for 6 hr at 50* then washed with NaCI solution on a Bfichner funnel to remove hydrazine. The NaC1 solution is displaced with the borate buffer and the hydrazide gel is kept in suspension at 0-4". Perchloric Acid Extraction of Keto Acids from Biological Samples Reagents
Perchloric acid, 2.9 M Potassium hydroxide, 3.6 M Universal indicator, commercial preparation
22
ANALYTICAL AND SYNTHETIC METHODS
[5]
Blood or plasma (0.5 ml) is mixed with water (0.5 ml) and ice-cold perchloric acid solution (0.5 ml) is added. After standing on ice for 15 min, the protein precipitate is removed by centrifugation at 10,000 g for 10 rain. The acid extract is neutralized with potassium hydroxide and 25/~1 of Universal indicator. Potassium perchlorate is removed by centrifugation. The extract can be stored at - 2 0 " or used immediately.
Purification and Derivatization of the 2-Keto Acids Reagents
Hydrazide gel suspended in borate buffer (see above) Acetic acid, 0.1 M Sodium chloride (AR), 0.1 M o-Phenylenediamine, 2 mg of the dihydrochloride per ml in 2 M HC1 containing 0.05% (v/v) 2-mercaptoethanol N 2 gas (O2-free) Sodium dithionite, solid (AR) Sodium sulfate, saturated aqueous solution Sodium sulfate, anhydrous (AR) 2-Ketooctanoic acid, 50 nM The procedure that follows is essentially that of Hayashi et al. 9 as modified by Livesey and Edwards. 1° To the extract from plasma or blood (1.0 ml) is added the 2-ketooctanoic acid (internal standard) (0.5 ml), acetic acid (1.0 ml), and sodium chloride (3 ml). The mixture is transferred to a glass column (150 × 5 mm) containing settled hydrazide gel (0.3 ml) supported on a small pad of glass wool. After the solution has filtered through the gel, it is washed with sodium chloride (5 ml). The gel is transferred to a screw-capped Sovirel tube (160 X 16 ram) and mixed with o-phenylenediamine solution (2.0 ml). The mixture is gassed with N2 (oxygen-free) and sodium dithionite (1-2 nag) is added to remove residual oxygen 3- 5 sec before cessation of gassing. The tube is sealed, heated at 80 ° for 2.0 hr, then cooled with cold water. Saturated sodium sulfate (4 ml) and ethyl acetate (5 ml) are added and the quinoxalinols extracted into the upper, organic phase by shaking for 5 min. This phase is removed and dried with anhydrous sodium sulfate (100 rag), overnight at 1-4*. This solution is taken to dryness on a rotary or vortex evaporator at 30". If not used immediately, the residue may be stored for a short time at room temperature in a desiccator.
[5]
QUANTITATIVE HPLC OF ~ T O ACIDS
23
High-Performance Liquid Chromatography
Conditions Column: 5/tm Lichrosorb RP8, 250 mm × 4.6 mm o.d. fitted with a silica precolumn (HPLC Technology Ltd) Liquid chromatograph operating conditions: Flow rate 1.5 ml/min; injection volume 20/~1 (Rheodyne valve injector); fluorescence detector set at excitation and emission wavelengths of 322 and 391 nm, respectively; temperature, 50 ° Mobile phase: Solution A, acetonitrile:water (4: 1, v/v); solution B, acetonitrile: water: 0.1 M Na2HPO4 adjusted to pH 7.0 with NaOH (1: 12:7, v/v/v) The data in the present paper were obtained using equipment from Perkin-Elmer Ltd, which comprised a Model 3B pump, LC-100 oven, and a Model 3000 fluorescence detector. On-line degassing of the mobile phase was used to reduce the possibility of quenching of the fluorescence by dissolved oxygen. The silica precolumn was used to saturate the mobile phase with silica. Data were processed by a Pye Unicam Ltd. data control center. The optimum excitation and emission wavelengths given were obtained by scanning the sample in the detector cell under stopped-flow conditions. Procedure The dried quinoxalinols are dissolved in dimethylformamide (40 gl) and water (100 #l) and aliquots (20/zl) injected onto the column. A concave gradient (Perkin-Elmer Code 2) is used to change the mobile-phase composition from 20 to 80% solution A over 35 rain for plasma or whole blood extracts. The column is equilibrated for l0 min with 20% solution A before injection of the next sample. Performance of the Method Variations in retention time for the three keto acids and in their peak area ratios relative to the internal standard are given in Table I and indicate the satisfactory reproducibility of the method. Plots of the ratio of peak area of keto acid versus keto acid concentration over the range 10 to 60 nmol relative to that of the internal standard (25 nmol) are linear and intercepts on the peak area axis are close to zero ( _+ 0.04). Relative standard deviations (RSD) for the slopes of these curves
24
ANALYTICAL AND SYNTHETIC METHODS
[5]
TABLE I REPRODUCIBILITY OF RETENTION TIME AND PEAK AREA RATIOS
Retention times (min) Keto acid
Range
Mean
Peak area ratio _+ RSD (%)
3-Methyl-2-ketobutyrate 4-Methyl-2-ketovalerate 3-Methyl-2-ketovalerate 2-Ketooctanoate
12.9-14.5 15.5-16.7 18.0-19.2 27.5-28.4
13.7 15.9 18.4 27.8
1.19 __- 2.3 1.25 ___0.9 1.19 -----2.4 --
for 3-methyl-2-ketobutyrate, 4-methyl-2-ketovalerate, and 3-methyl-2-ketovalerate are 1.2, 1.0, and 1.3%, respectively. Typical chromatograms of the keto acids alone and those extracted from plasma and whole blood are shown in Figs. 1, 2, and 3, respectively. The peaks of interest are well resolved and adequate for quantification. The overall performance of the method has been determined by taking blood or plasma spiked with known amounts of each acid through the 2
II
0 20 4Omin FIG. I. HPLC chromatogram of quinoxalinols of a standard mixture of keto adds. Peak I, 3-methyl-2-ketobutyrate; peak 2, 4-methyl-2-ketovalerate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-ketooctanoate (internal standard).
[5]
QUANTITATIVE H P L C OF KETO ACIDS
25
3
I J
0
20
40rain
FIG. 2. HPLC chmmatogram of quinoxalinols of the branched-chain 2-keto acids from normal human plasma. Peak 1, 3-methyl-2-ketobutyrate; peak 2, 4-methyl-2-ketovalerate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-kctooctanoate (internal standard).
whole procedure and calculating the amount recovered. Mean recovery values for the three acids were 111% (RSD 2.8%), 105% (RSD 6.3%), and 98% (RSD 2.8%), respectively. Similar experiments with whole blood gave values of 97% (RSD 1.3%), 89% (RSD 2.4%), and 78% (RSD 3.6%), respectively. Recovery values are known to be influenced by the purity of the individual acid,~ and the analyst is advised to determine these recovery values as frequently as possible as a check on the method. Lower recovery values for 3-methyl-2-ketobutyrate and 4-methyl-2-ketovalerate in whole blood have been noted in the enzymatic method where loss of keto acid with the protein precipitate was held to be responsible. The reproducibility of the procedures has been determined using sampies of plasma and whole blood and has been found to be quite satisfactory. Replicate analyses of plasma samples containing 13.4, 33.9, and 18.7 nmol/ml of the three acids gave RSD values of 1.4, 1.7, and 2.5%, respectively. For whole blood containing 9.5, 18.7, and 11.2 nmol/ml, the values were 2.1, 1.9, and 1.3%.
26
ANALYTICAL AND SYNTHETIC METHODS
[5]
4
A
0 20 40min FIG. 3. HPLC chromatogram of quinoxalinols of the branched-chain 2-keto acids from normal human whole blood. Peak 1, 3-mcthyl-2-ketobutyrat¢; peak 2, 4-methyl-2-ketovaicrate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-ketooctanoat¢ (internal standard).
Sensitivity, Precision, and Accuracy This procedure is extremely sensitiveand can detect as low as 0. I nmol of the kcto acid. Thc precision of the assay is within + 0.6 nmol/ml for 0.5 ml of normal h u m a n plasma. The accuracy of the method for plasma is within ___1 nmol/ml and corresponding values for normal h u m a n whole blood arc _ 0.3 and __.0.5 nmol/ml, respectively. Comments
on the Procedure
Standard solutions of the branched-chain 2-keto acids kept at 4 ° are more stable than that of the internal standard, but all appear to be stable for at least 2 months when frozen at neutral pH. The dedvatization procedure is sensitive to oxygen, ~° and results in a marked decrease for the ratio of 3-methyl-2-ketobutyrate relative to the internal standard and increases for 3-methyl-2-ketovalerate and 4-methyl-2-ketovalerate. The absence of oxygen therefore improves the performance of the method and markedly increases the sensitivity of the method for 3-methyl-2-ketobutyrate. The
[6]
GC-MS
ASSAY OF
BCH A N D BCK ACIDS
27
quinoxalinols are stable for at least 5 days at room temperature; after 7 days there is a decrease in the ratio of the peak area of the internal standard relative to that of each of the three keto acids. This procedure uses peak areas for quantification. If preferred, it is possible to use peak heights and obtain similar precision and accuracy. Plots of keto acid concentration calculated on peak height compared to those using peak area in 26 analyses of plasma and 26 analyses of whole blood gave linear relationships (passing close to the zero intercept, standard deviation 0.2 nmol/ml) with slopes of 1.03 (RSD 2.0%), 0.99 (2.5%), and 1.01 (0.9%), respectively, for the three acids. Reference Ranges One milliliter of human antecubital venous plasma collected after overnight fasting contains 40-80 (mean 63, RSD 25%) nmol of total branched-chain 2-keto acids, 8-16 (mean 13, RSD 22%) nmol of 3methyl-2-ketobutyrate, 20-40 (mean 31, RSD 25%) nmol of 4-methyl-2ketovalerate, and 10-30 (mean 19, RSD 32%) nmol of 3-methyl-2-ketovalerate. One milliliter of whole blood contains approximately 60% of the plasma values.
[6] D e t e r m i n a t i o n o f B r a n c h e d - C h a i n 2 - H y d r o x y a n d 2-Keto Acids by Mass Spectrometry By ORVAL A. MAMER and JANE A. MONTGOMERY
Introduction 2-Ketoisovaleric (KIVA), 2-ketoisocaproic (KICA), and 2-keto-3methyl-valeric (KMVA) acids are the transamination products of valine, leucine, and isoleucine, respectively, and are substrates for the branchedchain keto acid dehydrogenase enzyme complex found principally in liver and kidney. These acids accumulate in the serum and urine of acute patients with inherited errors of this complex in quantifies large enough to be found easily by coupled gas chromatography-mass spectrometry (GCMS). These acids are also of interest, for example, in fasting studies of the chronically obese, where one encounters concentrations of these acids more closely comparable with those for normal individuals. Largely unrecognized is the importance of the concentrations of the corresponding 2-hydroxyisovaleric (HIVA), 2-hydroxyisocaproic (HICA), and 2-hydroxyMETHODS IN ENZYMOLOGY, VOL. 166
Copyright© 1988by AcademicPress,Inc. All fightsof reproductionin any form r--~rved.
ANALYTICAL
AND SYNTHETIC
METHODS
[5]
[5] Quantitative High-Performance Liquid Chromatographic Analysis of Branched-Chain 2-Keto Acids in Biological Samples By ANGELA L. GASKING, WILLIAM T. E. EDWARDS, ANTHONY HOBSON-FROHOCK,MARINOS ELIA, and GEOFFREYLIVESEY The concentrations o f the branched-chain 2-keto acids, 4-methyl-2-ketovaleric acid (ketoleucine), 3-methyl-2-ketovaleric acid (ketoisoleucine), and 3-methyl-2-ketobutyric acid (ketovaline), in m a m m a l i a n tissues are small, |-3 making quantification difficult. The major tissue pools o f keto acids in the rat are plasma (and whole blood) and muscle. 2,3 In h u m a n blood the keto acids predominate in p l a s m # and partly in association with albumin. 5 H u m a n urine normally contains amounts o f these keto acids which are too small to quantify with satisfactory accuracy. Analysis o f the branched-chain 2-keto acids is important in biochemistry and clinical chemistry with concentrations in blood or plasma being elevated by starvation and diabetes 4 and several disorders o f branched-chain amino acid metabolism. 6 Several analytical methods have been developed to quantify the branched-chain 2-keto acids using gas chromatography, ~,7 high-performance liquid chromatography, s- 10 or enzymatic determination. 2 The enzymatic m e t h o d is rapid and reliable and sufficiently sensitive for analysis o f the keto acids in muscle, plasma, and whole blood, but does not distinguish between the three individual acids. A highly sensitive high-performance liquid chromatographic method is described below involving a single derivatization procedure to the stable quinoxalinols which are detected by fluorescence. The method is based on that o f Hayashi and co-workers, 8,9 with a modification to exclude oxygen during derivatization. 1° See also P. L. Crowell, R. H. Miller, and A. E. Harper, this volume [7]. 2G. Liveseyand P. Lund, Biochem. J. 188, 705 (1980);see also G. Liveseyand P. Lund, this volume [ 1]. 3S. M. Hutson and A. E. Harper, Am. J. Clin. Nutr. 34,, 173 (!981). 4 M. Elia and G. Livesey,Clin. Sci. 64, 517 (1983). 5G. Liveseyand P. Lund~Biochem. J. 212, 655 (1983). K. Tanaka and L. E. Rosenberg, in "The Metabolic Basis of Inherited Diseases" (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredriekson,J. L. Goldstein, and M. S. Brown, eds.), p. 440. McGraw-Hill,New York, 1983. 7T. C. Cree, S. M. Hutson, and A. E. Harper, Anal Biochem. 92, 156 (1979). a T. Hayashi, T. Hironori, H. Todoriki, and H. Naruse, AnaL Biochem. 122, 173 (1982). 9 T. Hayashi, H. Tsuehiya, and H. Naruse, J. Chromatogr. 273, 245 (1983). l0 G. Liveseyand W. T. E. Edwards, J. Chromatogr. 337, 98 (1985).
METHODS IN ENZYMOLOGY, VOL. 166
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
QUANTITATIVE H P L C OF KETO ACIDS
21
Principles of the Method The proteins of blood and plasma are precipitated with acid and the solution is purified by retention of the keto acids on a hydrazide gel. The hydrazones are converted anaerobically to the quinoxalinols with ophenylenediamine which are then separated and quantified by HPLC using a reversed-phase column and a fluorescence detector. Gel - CONH- NH 2 + R. CO" CO2H--*Gel- CONHN:CR- CO2H OH GeI-CONHN:CR.CO2H + ~
~
~ GeI-CONH.NH 2 +
~ N H 2
N
R
4-Methyl-2-ketovaleric acid [R = --CH2CH2CH(CH3) 2 3-Methyl-2-ketovaleric acid [R = --CH2CH(CH3)CH2CH3] 3-Methyl-2-ketobutyric acid [R = --CH2CH(CH3)2]
Preparation of the Hydrazide Gel Reagents
BioGel P-60, 100-200 mesh (Bio-Rad Ltd) Hydrazine hydrate, 98% Sodium chloride, 0.1 M Borate buffer, 0.1 M H2BO3 containing 0.2 M NaC1, 0.02 M disodium ethylenediaminetetraacetic acid (Na2 EDTA), 5 #M pentachlorophenol (final concentrations) to pH 7.3 with NaOH 'The preparative procedure is based on the method of Hayashi et al. 9 Dry gel (15 g) is added to water (200 ml) in a siliconized flask and left overnight. The gel suspension is heated to 50 ° and mixed with 80 ml of the hydrazine hydrate also at 50 °. The mixture is stirred for 6 hr at 50* then washed with NaCI solution on a Bfichner funnel to remove hydrazine. The NaC1 solution is displaced with the borate buffer and the hydrazide gel is kept in suspension at 0-4". Perchloric Acid Extraction of Keto Acids from Biological Samples Reagents
Perchloric acid, 2.9 M Potassium hydroxide, 3.6 M Universal indicator, commercial preparation
22
ANALYTICAL AND SYNTHETIC METHODS
[5]
Blood or plasma (0.5 ml) is mixed with water (0.5 ml) and ice-cold perchloric acid solution (0.5 ml) is added. After standing on ice for 15 min, the protein precipitate is removed by centrifugation at 10,000 g for 10 rain. The acid extract is neutralized with potassium hydroxide and 25/~1 of Universal indicator. Potassium perchlorate is removed by centrifugation. The extract can be stored at - 2 0 " or used immediately.
Purification and Derivatization of the 2-Keto Acids Reagents
Hydrazide gel suspended in borate buffer (see above) Acetic acid, 0.1 M Sodium chloride (AR), 0.1 M o-Phenylenediamine, 2 mg of the dihydrochloride per ml in 2 M HC1 containing 0.05% (v/v) 2-mercaptoethanol N 2 gas (O2-free) Sodium dithionite, solid (AR) Sodium sulfate, saturated aqueous solution Sodium sulfate, anhydrous (AR) 2-Ketooctanoic acid, 50 nM The procedure that follows is essentially that of Hayashi et al. 9 as modified by Livesey and Edwards. 1° To the extract from plasma or blood (1.0 ml) is added the 2-ketooctanoic acid (internal standard) (0.5 ml), acetic acid (1.0 ml), and sodium chloride (3 ml). The mixture is transferred to a glass column (150 × 5 mm) containing settled hydrazide gel (0.3 ml) supported on a small pad of glass wool. After the solution has filtered through the gel, it is washed with sodium chloride (5 ml). The gel is transferred to a screw-capped Sovirel tube (160 X 16 ram) and mixed with o-phenylenediamine solution (2.0 ml). The mixture is gassed with N2 (oxygen-free) and sodium dithionite (1-2 nag) is added to remove residual oxygen 3- 5 sec before cessation of gassing. The tube is sealed, heated at 80 ° for 2.0 hr, then cooled with cold water. Saturated sodium sulfate (4 ml) and ethyl acetate (5 ml) are added and the quinoxalinols extracted into the upper, organic phase by shaking for 5 min. This phase is removed and dried with anhydrous sodium sulfate (100 rag), overnight at 1-4*. This solution is taken to dryness on a rotary or vortex evaporator at 30". If not used immediately, the residue may be stored for a short time at room temperature in a desiccator.
[5]
QUANTITATIVE HPLC OF ~ T O ACIDS
23
High-Performance Liquid Chromatography
Conditions Column: 5/tm Lichrosorb RP8, 250 mm × 4.6 mm o.d. fitted with a silica precolumn (HPLC Technology Ltd) Liquid chromatograph operating conditions: Flow rate 1.5 ml/min; injection volume 20/~1 (Rheodyne valve injector); fluorescence detector set at excitation and emission wavelengths of 322 and 391 nm, respectively; temperature, 50 ° Mobile phase: Solution A, acetonitrile:water (4: 1, v/v); solution B, acetonitrile: water: 0.1 M Na2HPO4 adjusted to pH 7.0 with NaOH (1: 12:7, v/v/v) The data in the present paper were obtained using equipment from Perkin-Elmer Ltd, which comprised a Model 3B pump, LC-100 oven, and a Model 3000 fluorescence detector. On-line degassing of the mobile phase was used to reduce the possibility of quenching of the fluorescence by dissolved oxygen. The silica precolumn was used to saturate the mobile phase with silica. Data were processed by a Pye Unicam Ltd. data control center. The optimum excitation and emission wavelengths given were obtained by scanning the sample in the detector cell under stopped-flow conditions. Procedure The dried quinoxalinols are dissolved in dimethylformamide (40 gl) and water (100 #l) and aliquots (20/zl) injected onto the column. A concave gradient (Perkin-Elmer Code 2) is used to change the mobile-phase composition from 20 to 80% solution A over 35 rain for plasma or whole blood extracts. The column is equilibrated for l0 min with 20% solution A before injection of the next sample. Performance of the Method Variations in retention time for the three keto acids and in their peak area ratios relative to the internal standard are given in Table I and indicate the satisfactory reproducibility of the method. Plots of the ratio of peak area of keto acid versus keto acid concentration over the range 10 to 60 nmol relative to that of the internal standard (25 nmol) are linear and intercepts on the peak area axis are close to zero ( _+ 0.04). Relative standard deviations (RSD) for the slopes of these curves
24
ANALYTICAL AND SYNTHETIC METHODS
[5]
TABLE I REPRODUCIBILITY OF RETENTION TIME AND PEAK AREA RATIOS
Retention times (min) Keto acid
Range
Mean
Peak area ratio _+ RSD (%)
3-Methyl-2-ketobutyrate 4-Methyl-2-ketovalerate 3-Methyl-2-ketovalerate 2-Ketooctanoate
12.9-14.5 15.5-16.7 18.0-19.2 27.5-28.4
13.7 15.9 18.4 27.8
1.19 __- 2.3 1.25 ___0.9 1.19 -----2.4 --
for 3-methyl-2-ketobutyrate, 4-methyl-2-ketovalerate, and 3-methyl-2-ketovalerate are 1.2, 1.0, and 1.3%, respectively. Typical chromatograms of the keto acids alone and those extracted from plasma and whole blood are shown in Figs. 1, 2, and 3, respectively. The peaks of interest are well resolved and adequate for quantification. The overall performance of the method has been determined by taking blood or plasma spiked with known amounts of each acid through the 2
II
0 20 4Omin FIG. I. HPLC chromatogram of quinoxalinols of a standard mixture of keto adds. Peak I, 3-methyl-2-ketobutyrate; peak 2, 4-methyl-2-ketovalerate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-ketooctanoate (internal standard).
[5]
QUANTITATIVE H P L C OF KETO ACIDS
25
3
I J
0
20
40rain
FIG. 2. HPLC chmmatogram of quinoxalinols of the branched-chain 2-keto acids from normal human plasma. Peak 1, 3-methyl-2-ketobutyrate; peak 2, 4-methyl-2-ketovalerate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-kctooctanoate (internal standard).
whole procedure and calculating the amount recovered. Mean recovery values for the three acids were 111% (RSD 2.8%), 105% (RSD 6.3%), and 98% (RSD 2.8%), respectively. Similar experiments with whole blood gave values of 97% (RSD 1.3%), 89% (RSD 2.4%), and 78% (RSD 3.6%), respectively. Recovery values are known to be influenced by the purity of the individual acid,~ and the analyst is advised to determine these recovery values as frequently as possible as a check on the method. Lower recovery values for 3-methyl-2-ketobutyrate and 4-methyl-2-ketovalerate in whole blood have been noted in the enzymatic method where loss of keto acid with the protein precipitate was held to be responsible. The reproducibility of the procedures has been determined using sampies of plasma and whole blood and has been found to be quite satisfactory. Replicate analyses of plasma samples containing 13.4, 33.9, and 18.7 nmol/ml of the three acids gave RSD values of 1.4, 1.7, and 2.5%, respectively. For whole blood containing 9.5, 18.7, and 11.2 nmol/ml, the values were 2.1, 1.9, and 1.3%.
26
ANALYTICAL AND SYNTHETIC METHODS
[5]
4
A
0 20 40min FIG. 3. HPLC chromatogram of quinoxalinols of the branched-chain 2-keto acids from normal human whole blood. Peak 1, 3-mcthyl-2-ketobutyrat¢; peak 2, 4-methyl-2-ketovaicrate; peak 3, 3-methyl-2-ketovalerate; peak 4, 2-ketooctanoat¢ (internal standard).
Sensitivity, Precision, and Accuracy This procedure is extremely sensitiveand can detect as low as 0. I nmol of the kcto acid. Thc precision of the assay is within + 0.6 nmol/ml for 0.5 ml of normal h u m a n plasma. The accuracy of the method for plasma is within ___1 nmol/ml and corresponding values for normal h u m a n whole blood arc _ 0.3 and __.0.5 nmol/ml, respectively. Comments
on the Procedure
Standard solutions of the branched-chain 2-keto acids kept at 4 ° are more stable than that of the internal standard, but all appear to be stable for at least 2 months when frozen at neutral pH. The dedvatization procedure is sensitive to oxygen, ~° and results in a marked decrease for the ratio of 3-methyl-2-ketobutyrate relative to the internal standard and increases for 3-methyl-2-ketovalerate and 4-methyl-2-ketovalerate. The absence of oxygen therefore improves the performance of the method and markedly increases the sensitivity of the method for 3-methyl-2-ketobutyrate. The
[6]
GC-MS
ASSAY OF
BCH A N D BCK ACIDS
27
quinoxalinols are stable for at least 5 days at room temperature; after 7 days there is a decrease in the ratio of the peak area of the internal standard relative to that of each of the three keto acids. This procedure uses peak areas for quantification. If preferred, it is possible to use peak heights and obtain similar precision and accuracy. Plots of keto acid concentration calculated on peak height compared to those using peak area in 26 analyses of plasma and 26 analyses of whole blood gave linear relationships (passing close to the zero intercept, standard deviation 0.2 nmol/ml) with slopes of 1.03 (RSD 2.0%), 0.99 (2.5%), and 1.01 (0.9%), respectively, for the three acids. Reference Ranges One milliliter of human antecubital venous plasma collected after overnight fasting contains 40-80 (mean 63, RSD 25%) nmol of total branched-chain 2-keto acids, 8-16 (mean 13, RSD 22%) nmol of 3methyl-2-ketobutyrate, 20-40 (mean 31, RSD 25%) nmol of 4-methyl-2ketovalerate, and 10-30 (mean 19, RSD 32%) nmol of 3-methyl-2-ketovalerate. One milliliter of whole blood contains approximately 60% of the plasma values.
[6] D e t e r m i n a t i o n o f B r a n c h e d - C h a i n 2 - H y d r o x y a n d 2-Keto Acids by Mass Spectrometry By ORVAL A. MAMER and JANE A. MONTGOMERY
Introduction 2-Ketoisovaleric (KIVA), 2-ketoisocaproic (KICA), and 2-keto-3methyl-valeric (KMVA) acids are the transamination products of valine, leucine, and isoleucine, respectively, and are substrates for the branchedchain keto acid dehydrogenase enzyme complex found principally in liver and kidney. These acids accumulate in the serum and urine of acute patients with inherited errors of this complex in quantifies large enough to be found easily by coupled gas chromatography-mass spectrometry (GCMS). These acids are also of interest, for example, in fasting studies of the chronically obese, where one encounters concentrations of these acids more closely comparable with those for normal individuals. Largely unrecognized is the importance of the concentrations of the corresponding 2-hydroxyisovaleric (HIVA), 2-hydroxyisocaproic (HICA), and 2-hydroxyMETHODS IN ENZYMOLOGY, VOL. 166
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