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A gas chromatographic method for the determination of acetone and acetoacetic acid in urine
Clinica Chmica
Acta, 133 (1983) 223-226
Brief technical
note
A gas chromatographic method for the determination of acetone and acetoacetic acid in urine Keiko Kobayashi
*, Masayuki
Okada, Yukihiko
Glfu College of Pharmacy, Mitahora,
(Received
Yasuda and Satoshi Kawai
Gifu 502 (Japan)
March 2nd; revision May 18th, 1983)
Introduction There are several types of reactions most commonly employed for calorimetric determination of ketone bodies in urine. Salicylaldehyde [ 1,2] and 2,4-dinitrophenylhydrazine [3,4] have been used for determination of acetone. Ferric chloride reacts with acetoacetic acid but not with acetone, and nitroprusside [5] also reacts sensitively with acetoacetic acid. The nitroprusside test was standardized and has now become available as a dip and read test called Ketostix, a registered trademark of the Ames Company Inc., Elkhart, IN, USA. The test detects 490 pmol/l of acetoacetate in urine, but is practically non-reactive to acetone. We have found pentafluorobenzyloxylammonium chloride (PFBOA) to be an excellent derivatizing agent in the gas chromatographic (GC) determination of low-molecular-weight carbonyl compounds such as aldehydes, ketones [6] and a-keto acids [7] in aqueous solution. We are not aware of a report dealing with a GC determination of ketone bodies. As an extension of our work on GC of carbonyl compounds, this paper describes a GC determination of acetone and acetoacetic acid in urine. Materials and methods Reagents PFBOA was obtained from Tokyo Kasei, Tokyo, Japan. Lithium acetoacetate was purchased from Sigma, St. Louis, MO, USA. p-Chlorobenzyl chloride and hexachlorobenzene were obtained from Wako Junyaku, Osaka, Japan and were used as internal standard. * All correspondence should be addressed to: Mitahora-higashi 5 chome, Gifu 502, Japan.
0009-8981/83/$03.00
Keiko
0 1983 Elsevier Science Publishers
Kobayashi,
B.V.
Gifu
College
of Pharmacy,
6-1,
224
Apparatus and conditions A Shimadzu Model GC 4BPF gas chromatograph equipped with a hydrogen flame-ionization detector (FJD) was used. Separations were carried out by using a glass column, 2.0 m x 3 mm id., packed with 3% XL60 on 80~100 mesh Celite 545(AW DMCS). The column temperature was 12O’C for acetone and was 180°C for acetoacetic acid. Procedure Acetone in urine. One ml of urine was placed in a lo-ml glass-stoppered test tube and diluted with 1 ml of water. In case of urine samples with an acetone concentration greater than 862 pmol/l, the urine was diluted with sufficient water to reduce the acetone concentration to a value below 862 pmol/l. To the sample solution was added 0.5 ml of PFBOA solution (20 mmol/l in water), and the mixture was allowed to stand for 60 min at room temperature. To the reaction mixture, saturated with sodium chloride, was added 1 drop of 9 mol./I sulfuric acid and the mixture was extracted with 0.3 ml of n-hexane containing 180 pg of p-chlorobenzyl chloride as internal standard. The excess sodium chloride and the aqueous layer were removed with the aid of a syringe with a long needle, a few grains of anhydrous sodium sulfate were added to dry the hexane extract and an aliquot was injected on to the GC column. Acetoacetic acid in urine. One ml of urine diluted, if necessary, as required for measurement, was treated in the same manner as for acetone in urine. After derivatization with PFBOA, the reaction mixture was extracted with 1 ml of hexane containing 250 pg of hexachlorobenzene as internal standard. The extract was transferred to a 3-ml pear-shaped flask and evaporated to dryness. An excess volume of freshly prepared diazomethane in diethyl ether was added to the residue and the mixture was allowed to stand for 10 min at room temperature to convert the oxime into its methyl ester [7], The reaction mixture was evaporated to dryness again and the residue was dissolved in 0.2 ml of hexane for GC analysis. Quantitation of acetone and acetoacetic acid was carried out using calibration graphs obtained from known amounts of acetone or acetoacetic acid, respectively. Results and discussion The reaction of carbonyl compounds with PFBOA proceeded readily in weakly acidic media (pH 2-5) at room temperature to yieid derivatives extractable from the aqueous solution with organic solvents. The resulting derivatives were very volatile, therefore the GC separation could be carried out at low temperatures. In this work, PFBOA was applied to the micro-determination of acetone and acetoacetic acid in urine. The O-pentafluorobenzyloximes were readily extracted with hexane from acid solution (pH 3). Because of these solubility characteristics it was possible to eliminate interference from co-existing substances in urine. Acetoacetic acid underwent rapid decomposition in acidic media to yield acetone [8]. Accordingly. a
225
standard solution of lithium acetoacetate was prepared. A solution of lithium acetoacetate (926 pmol/l) in urine was stable at least for a day in a refrigerator. Typical GC separations of acetone (A) and acetoacetic acid (B) in urine are A
0 2
_I 3
I
I
I
0
4
0,
8min
I
I
0
4
I,
0 min
Fig. 1. Gas chromatograms of ketone bodies in urine on a 2.0-m 3% XE-60 column, at 120°C (A) and 180°C (B). Peaks: 1, acetone 0-pentafluorobenzyloxime; 2, p-chlorobenzyl chloride; 3. methyl acetoacetate 0-pentafluorobenzyloxime; 4, hexachlorobenzene.
illustrated in Fig. 1. The peak of acetone or acetoacetic acid on the chromatogram corresponded to acetone 345 pmol/l or acetoacetic acid 490 pmol/l in urine, respectively. In the chromatograms of the O-pentafluorobenzyloxime of methyl acetoacetate, formation of a double peak was observed, corresponding to syn- and anti-isomer. The total area of the two peaks was used for determination of acetoacetic acid. A sample solution containing an individual amount of acetone or acetoacetic acid was measured according to the procedure described under ‘Methods’ and linear calibration graphs passing through the origin were obtained in the range 17-862 pmol/l of acetone and 20-980 pmol/l of acetoacetic acid added in IO-fold diluted urine. The reproducibilities of the methods were examined with an identical sample solution containing 345 pmol/l of acetone or 490 pmol/l of acetoacetic acid in urine and the coefficients of variation obtained were 1.7% (n = 5) for acetone and 2.00% (n = 5) for acetoacetic acid. In the method reported here, the absolute sensitivity of acetone was 3.45 pmol/l and that of acetoacetic acid was 10 pmol/l. They are about 100 times more sensitive than calorimetric methods reported in the Iiterature. Some urines from diabetics and patients with autointoxication were tested with both the proposed method and the Ketostix method, and the results are shown in Table I. The present method appears to have no advantage over Ketostix test with respect to rapidity and simplicity of technique. However, because of its greater specificity and sensitivity it may be preferred. The method gives a reliable determination of acetone and acetoacetic acid levels in the same sample and is superior to the calorimetric method which gives false-positive reactions. The method
226
TABLE
I
Data on urine from patients
with diabetic
Patient
Acetone
Normal
7* 5 4
Diabetic
or autointoxication
155 103 13 Ii 19 16
Autointoxication
7 793 534 4 207
11207 * All concentrations
expressed
Acetoacetic
acid
Ketostix
test
< 10 _
-c IO <: 10 333 147 c: 10 < 10 20 20
_ _ _ _
1667 196
++ _
2255 6373
++ +++
as pmol/l.
may also be further applied for the determination of ~-hydroxybutyric conversion to acetone by oxidation with acid-dichromate reagent 141.
acid after
Acknowledgement The authors samples.
thank
Dr. E. Asakura
and
Dr. E. Aoyama
for supplying
urine
References I Behre JA. A modified salicylaldehyde method for the determination of acetone bodies in blood and urine. J Biol Chem I940; 136: 25-34. 2 Reid RL. The deter~nation of ketone bodies in blood. Analyst 1960; 85: 265-271. 3 Greenberg LA, Lester D. A micromethod for the determination of acetone and ketone bodies. J Biol Chem 1944; 154: 177-190. 4 Mayes PA, Robson W. The determination of ketone bodies. Biochem J 1957; 67: 11-15. 5 Free HM, Smeby RR, Cook MH, Free AH. A comparative study of qualitative tests for ketones in urine and serum. Clin Chem 1958; 4: 323-330. 6 Kobayashi K. Tanaka M, Kawai S. Gas chromatographic determination of low-molecuiar-weight carbonyl compounds in aqueous solution as their ~-(2,3,4,5,6-pentafluoro~nzyl)oximes. J Chromatogr 1980; 187: 413-417. 7 Kobayashi K, Fukui E, Tanaka M, Kawai S. Gas chromatographic analysis of cu-keto acids in aqueous solution as the 0-(2,3,4,5,6-pentafluorobenzyl)oximes of their methyl esters. J Chromatogr 1980; 187: 93-98. 8 Widmark EMP. Kinetics of the ketonic decomposition of acetoacetic acid. Acta Med Stand 1920; 53: 393-421.
Acta, 133 (1983) 223-226
Brief technical
note
A gas chromatographic method for the determination of acetone and acetoacetic acid in urine Keiko Kobayashi
*, Masayuki
Okada, Yukihiko
Glfu College of Pharmacy, Mitahora,
(Received
Yasuda and Satoshi Kawai
Gifu 502 (Japan)
March 2nd; revision May 18th, 1983)
Introduction There are several types of reactions most commonly employed for calorimetric determination of ketone bodies in urine. Salicylaldehyde [ 1,2] and 2,4-dinitrophenylhydrazine [3,4] have been used for determination of acetone. Ferric chloride reacts with acetoacetic acid but not with acetone, and nitroprusside [5] also reacts sensitively with acetoacetic acid. The nitroprusside test was standardized and has now become available as a dip and read test called Ketostix, a registered trademark of the Ames Company Inc., Elkhart, IN, USA. The test detects 490 pmol/l of acetoacetate in urine, but is practically non-reactive to acetone. We have found pentafluorobenzyloxylammonium chloride (PFBOA) to be an excellent derivatizing agent in the gas chromatographic (GC) determination of low-molecular-weight carbonyl compounds such as aldehydes, ketones [6] and a-keto acids [7] in aqueous solution. We are not aware of a report dealing with a GC determination of ketone bodies. As an extension of our work on GC of carbonyl compounds, this paper describes a GC determination of acetone and acetoacetic acid in urine. Materials and methods Reagents PFBOA was obtained from Tokyo Kasei, Tokyo, Japan. Lithium acetoacetate was purchased from Sigma, St. Louis, MO, USA. p-Chlorobenzyl chloride and hexachlorobenzene were obtained from Wako Junyaku, Osaka, Japan and were used as internal standard. * All correspondence should be addressed to: Mitahora-higashi 5 chome, Gifu 502, Japan.
0009-8981/83/$03.00
Keiko
0 1983 Elsevier Science Publishers
Kobayashi,
B.V.
Gifu
College
of Pharmacy,
6-1,
224
Apparatus and conditions A Shimadzu Model GC 4BPF gas chromatograph equipped with a hydrogen flame-ionization detector (FJD) was used. Separations were carried out by using a glass column, 2.0 m x 3 mm id., packed with 3% XL60 on 80~100 mesh Celite 545(AW DMCS). The column temperature was 12O’C for acetone and was 180°C for acetoacetic acid. Procedure Acetone in urine. One ml of urine was placed in a lo-ml glass-stoppered test tube and diluted with 1 ml of water. In case of urine samples with an acetone concentration greater than 862 pmol/l, the urine was diluted with sufficient water to reduce the acetone concentration to a value below 862 pmol/l. To the sample solution was added 0.5 ml of PFBOA solution (20 mmol/l in water), and the mixture was allowed to stand for 60 min at room temperature. To the reaction mixture, saturated with sodium chloride, was added 1 drop of 9 mol./I sulfuric acid and the mixture was extracted with 0.3 ml of n-hexane containing 180 pg of p-chlorobenzyl chloride as internal standard. The excess sodium chloride and the aqueous layer were removed with the aid of a syringe with a long needle, a few grains of anhydrous sodium sulfate were added to dry the hexane extract and an aliquot was injected on to the GC column. Acetoacetic acid in urine. One ml of urine diluted, if necessary, as required for measurement, was treated in the same manner as for acetone in urine. After derivatization with PFBOA, the reaction mixture was extracted with 1 ml of hexane containing 250 pg of hexachlorobenzene as internal standard. The extract was transferred to a 3-ml pear-shaped flask and evaporated to dryness. An excess volume of freshly prepared diazomethane in diethyl ether was added to the residue and the mixture was allowed to stand for 10 min at room temperature to convert the oxime into its methyl ester [7], The reaction mixture was evaporated to dryness again and the residue was dissolved in 0.2 ml of hexane for GC analysis. Quantitation of acetone and acetoacetic acid was carried out using calibration graphs obtained from known amounts of acetone or acetoacetic acid, respectively. Results and discussion The reaction of carbonyl compounds with PFBOA proceeded readily in weakly acidic media (pH 2-5) at room temperature to yieid derivatives extractable from the aqueous solution with organic solvents. The resulting derivatives were very volatile, therefore the GC separation could be carried out at low temperatures. In this work, PFBOA was applied to the micro-determination of acetone and acetoacetic acid in urine. The O-pentafluorobenzyloximes were readily extracted with hexane from acid solution (pH 3). Because of these solubility characteristics it was possible to eliminate interference from co-existing substances in urine. Acetoacetic acid underwent rapid decomposition in acidic media to yield acetone [8]. Accordingly. a
225
standard solution of lithium acetoacetate was prepared. A solution of lithium acetoacetate (926 pmol/l) in urine was stable at least for a day in a refrigerator. Typical GC separations of acetone (A) and acetoacetic acid (B) in urine are A
0 2
_I 3
I
I
I
0
4
0,
8min
I
I
0
4
I,
0 min
Fig. 1. Gas chromatograms of ketone bodies in urine on a 2.0-m 3% XE-60 column, at 120°C (A) and 180°C (B). Peaks: 1, acetone 0-pentafluorobenzyloxime; 2, p-chlorobenzyl chloride; 3. methyl acetoacetate 0-pentafluorobenzyloxime; 4, hexachlorobenzene.
illustrated in Fig. 1. The peak of acetone or acetoacetic acid on the chromatogram corresponded to acetone 345 pmol/l or acetoacetic acid 490 pmol/l in urine, respectively. In the chromatograms of the O-pentafluorobenzyloxime of methyl acetoacetate, formation of a double peak was observed, corresponding to syn- and anti-isomer. The total area of the two peaks was used for determination of acetoacetic acid. A sample solution containing an individual amount of acetone or acetoacetic acid was measured according to the procedure described under ‘Methods’ and linear calibration graphs passing through the origin were obtained in the range 17-862 pmol/l of acetone and 20-980 pmol/l of acetoacetic acid added in IO-fold diluted urine. The reproducibilities of the methods were examined with an identical sample solution containing 345 pmol/l of acetone or 490 pmol/l of acetoacetic acid in urine and the coefficients of variation obtained were 1.7% (n = 5) for acetone and 2.00% (n = 5) for acetoacetic acid. In the method reported here, the absolute sensitivity of acetone was 3.45 pmol/l and that of acetoacetic acid was 10 pmol/l. They are about 100 times more sensitive than calorimetric methods reported in the Iiterature. Some urines from diabetics and patients with autointoxication were tested with both the proposed method and the Ketostix method, and the results are shown in Table I. The present method appears to have no advantage over Ketostix test with respect to rapidity and simplicity of technique. However, because of its greater specificity and sensitivity it may be preferred. The method gives a reliable determination of acetone and acetoacetic acid levels in the same sample and is superior to the calorimetric method which gives false-positive reactions. The method
226
TABLE
I
Data on urine from patients
with diabetic
Patient
Acetone
Normal
7* 5 4
Diabetic
or autointoxication
155 103 13 Ii 19 16
Autointoxication
7 793 534 4 207
11207 * All concentrations
expressed
Acetoacetic
acid
Ketostix
test
< 10 _
-c IO <: 10 333 147 c: 10 < 10 20 20
_ _ _ _
1667 196
++ _
2255 6373
++ +++
as pmol/l.
may also be further applied for the determination of ~-hydroxybutyric conversion to acetone by oxidation with acid-dichromate reagent 141.
acid after
Acknowledgement The authors samples.
thank
Dr. E. Asakura
and
Dr. E. Aoyama
for supplying
urine
References I Behre JA. A modified salicylaldehyde method for the determination of acetone bodies in blood and urine. J Biol Chem I940; 136: 25-34. 2 Reid RL. The deter~nation of ketone bodies in blood. Analyst 1960; 85: 265-271. 3 Greenberg LA, Lester D. A micromethod for the determination of acetone and ketone bodies. J Biol Chem 1944; 154: 177-190. 4 Mayes PA, Robson W. The determination of ketone bodies. Biochem J 1957; 67: 11-15. 5 Free HM, Smeby RR, Cook MH, Free AH. A comparative study of qualitative tests for ketones in urine and serum. Clin Chem 1958; 4: 323-330. 6 Kobayashi K. Tanaka M, Kawai S. Gas chromatographic determination of low-molecuiar-weight carbonyl compounds in aqueous solution as their ~-(2,3,4,5,6-pentafluoro~nzyl)oximes. J Chromatogr 1980; 187: 413-417. 7 Kobayashi K, Fukui E, Tanaka M, Kawai S. Gas chromatographic analysis of cu-keto acids in aqueous solution as the 0-(2,3,4,5,6-pentafluorobenzyl)oximes of their methyl esters. J Chromatogr 1980; 187: 93-98. 8 Widmark EMP. Kinetics of the ketonic decomposition of acetoacetic acid. Acta Med Stand 1920; 53: 393-421.