Article Acetone 2

Hindawi Publishing Corporation
Journal of Analytical Methods in Chemistry

Volume 2016, Article ID 5176320, 7 pages
http://dx.doi.org/10.1155/2016/5176320
Research Article
Quantitative Clinical Diagnostic Analysis of Acetone in Human
Blood by HPLC: A Metabolomic Search for Acetone as Indicator
Esin Akgul Kalkan,1 Mehtap Sahiner,2 Dilek Ulker Cakir,3

Duygu Alpaslan,4 and Selehattin Yilmaz5
1
Department of Forensic Medicine, Faculty of Medicine, Canakkale Onsekiz Mart University,
Terzioglu Campus, 17020 Canakkale, Turkey
2
Department of Leather Engineering, Faculty of Engineering, Ege University, Bornova, 35100 İzmir, Turkey
3
Department of Clinical Biochemistry, Faculty of Medicine, Canakkale Onsekiz Mart University, Terzioglu Campus,
17020 Canakkale, Turkey
4
Department of Chemical Engineering, Faculty of Engineering, Yuzuncu Yil University, 65080 Van, Turkey
5
Department of Chemistry, Faculty of Sciences and Arts, Canakkale Onsekiz Mart University, Terzioglu Campus,
17020 Canakkale, Turkey
Correspondence should be addressed to Selehattin Yilmaz; [email protected]
Received 13 February 2016; Revised 26 April 2016; Accepted 4 May 2016
Academic Editor: Mohamed Abdel-Rehim
Copyright © 2016 Esin Akgul Kalkan et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Using high-performance liquid chromatography (HPLC) and 2,4-dinitrophenylhydrazine (2,4-DNPH) as a derivatizing reagent,
an analytical method was developed for the quantitative determination of acetone in human blood. The determination was carried
out at 365 nm using an ultraviolet-visible (UV-Vis) diode array detector (DAD). For acetone as its 2,4-dinitrophenylhydrazone
derivative, a good separation was achieved with a ThermoAcclaim C18 column (15 cm × 4.6 mm × 3 𝜇m) at retention time (𝑡R )
12.10 min and flowrate of 1 mL min−1 using a (methanol/acetonitrile) water elution gradient. The methodology is simple, rapid,
sensitive, and of low cost, exhibits good reproducibility, and allows the analysis of acetone in biological fluids. A calibration
curve was obtained for acetone using its standard solutions in acetonitrile. Quantitative analysis of acetone in human blood was
successfully carried out using this calibration graph. The applied method was validated in parameters of linearity, limit of detection
and quantification, accuracy, and precision. We also present acetone as a useful tool for the HPLC-based metabolomic investigation
of endogenous metabolism and quantitative clinical diagnostic analysis.
1. Introduction alcohol ingestions, drug toxicities, inborn errors of ketone

metabolism, alcoholic ketoacidosis, delirium tremens, and
Acetone is the simplest ketone compound. In general, acetone
is not considered harmful, and the World Health Organi- hypothermia [6–9]. DM is a disease involving environmental
zation has not classified acetone as carcinogenic. However, and genetic factors. The main symptom of DM is a high
its prolonged inhalation can not only cause irritation of blood glucose concentration depending on insulin deficiency.
the mucous membranes, headaches, confusion, and narcotic In this case the body cannot fully use glucose but could
effects, but lead to coma as well [1–5]. use fatty metabolism instead of glucose for energy [9].
For etiological reasons, acetonaemia is classified to be DM, especially diabetes and autoimmune associated diseases,
of endogenous and exogenous origin [6, 7]. Multiple tox- thyroid disease, and diseases that can accompany diabetes
icities and physiopathological conditions result in keto- (hypertension, cardiovascular disease, cerebrovascular dis-
sis (acetonaemia particularly), including diabetes mellitus ease, and renal insufficiency) can cause pathological changes
(DM), starvation coupled with physiologic stress, prolonged in most of the tissues, organs, and biological fluids depending
exercise, during pregnancy, and ethanol toxicity, other on lipotoxicity and glucotoxicity.
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2 Journal of Analytical Methods in Chemistry
A lot of medical, chemical, and medicolegal investigations Cobas 6000 (Roche, Germany) autoanalyzer was used for
have been carried out with the determination of acetone blood glucose levels. Qualitative analysis of total ketones
in blood and other biological fluids [2, 6, 7, 10]. Over the in urine was evaluated by Iris Iricel 2000 Analyzer (Icem
decades, several methods have been used for its determi- Velocity).
nation in biological samples. In the beginning, colorimetric
methods were developed and used for the determination of 2.3. Sample Collection and Procedure for the Determination
acetone in plasma [11–13]. These methods have common dis- of Acetone in Human Blood. In the first stage of our study,
advantages such as the lack of specificity and detection limit. following a 12-hour fasting venous blood samples were taken
In recent decades, gas chromatographs equipped with flame from patients admitted to hospital of the Faculty of Medicine,
ionization detectors or mass spectrometric detectors have Canakkale Onsekiz Mart University. The human blood and
been developed for determination of acetone concentrations urine samples were directly collected into a tube (without
in body fluids and in expired air [14–18]. Enzymatic methods a collection device) between 08:30 and 11:00 am. Clinical
are more specific but more complex and have long assay Biochemistry Laboratory blood and urine glucose tests were
times and gas chromatographic methods, although widely studied for routine biochemistry using a urine autoanalyzer.
used, are applied with difficulty as routine tests [2]. The Test results were screened for hyperglycemia and ketonuria.
determination of acetone in the blood is most important in The patients were divided into high blood glucose and urine
clinical diagnostic laboratory studies. There are three ketone ketone positive subjects (Group 1) and high blood glucose
bodies, while the two main ketone bodies are acetoacetate and urine ketone negative example subjects (Group 2). The
(AcAc) and 3-b-hydroxybutyrate (3HB), the third ketone patients with hyperglycemia were 8 females and 7 males (age:
body; acetone (Ac) is found minimum level [9]. Ketone 21–87; 𝑛 = 15), while 5 female and 2 male patients had positive
bodies are produced by the liver and used peripherally as an urine ketones (age: 21–68, 𝑛 = 7) and 5 male and 3 female
energy source when glucose is not readily available [9, 19]. patients had negative urine ketones (age: 55–87, 𝑛 = 8). The
Ketone bodies are three water-soluble compounds that are blood glucose levels varied between 110 and 320 mg/dL in our
produced as by-products when fatty acids are broken down patients (Table 1).
for energy in the liver and kidney [19]. Ketone bodies are pro- In the second stage of the study, to determine the probable
duced from acetyl-CoA mainly in the mitochondrial matrix positive acetone, its quantitative analysis was carried out
of hepatocytes when carbohydrates are so scarce that energy in biological fluids using the HPLC technique. The blood
must be obtained from breaking down fatty acids [9, 20]. samples were immediately prepared for HPLC analysis car-
Also, acetone is produced by spontaneous decarboxylation ried out within 8 hours after sample collection. A method
of acetoacetate [6–9]. Acetone cannot be converted back to for the determination of acetone in human blood by HPLC
acetyl-CoA, so it is excreted in the urine or exhaled [21]. was developed. Plasma specimens were deproteinized with
Recently, blood or urine testing kits have been used to test for acetonitrile (1 : 1, v/v); 2,4-DNPH is added to the supernatant
the presence of acetone in clinical biochemistry laboratories. (filtered blood samples) and treated with acetonitrile (2 : 1,
Acetone can also be quantified by sampling the human blood v/v) to prevent crystallization of the synthesized phenylhy-
and testing by gas chromatography [22]. Brega et al. described drazone. An aliquot (20 microliters) of the reaction mix-
a rapid and simple HPLC procedure that can be used for the ture was subjected to HPLC at ambient temperature using
routine measurement of acetone in biological fluids, such as ThermoAcclaim-C18 (15 cm × 4.6 mm × 3 𝜇m) column and
plasma and urine. According to Fujii et al., it is proposed that UV-Vis DAD with (methanol/acetonitrile) water as eluent
liquid chromatography with fluorescence (LC-FL) seems to at a flowrate of 1 mL min−1 and detection at 365 nm [2, 3].
be useful for the determination of acetone in the saliva [23]. The experimental procedures were conducted in accordance
In this paper, we present a rapid and simple HPLC with the ethical standards of the Helsinki Declaration and
technique using 2,4-DNPH as a derivatizing reagent for approved by the Canakkale Onsekiz Mart University Human
quantitative determination and metabolomic research of Research Ethics Committee. Written informed consent was
acetone in biological fluid such as human blood. obtained from all the subjects.
2. Material and Methods 2.4. Acetone Labeling and HPLC Analysis. The quantitative
analysis of acetone using HPLC was performed after labeling
2.1. Reagents and Standards. 2,4-DNPH (Sigma-Aldrich, with 2,4-DNPH. The 2,4-dinitrophenylhydrazone standards
97%), acetone (Merck, 99.8%), acetonitrile (Sigma-Aldrich, were prepared by mixing A and B solutions: (A): 0.40 g of 2,4-
99.8%), and methanol (Merck, 99.8%) were used in this study. DNPH dissolved in 2.00 mL of H2 SO4 + 3.00 mL of H2 O +
All other chemicals were of analytical reagent grade and used 10.0 mL ethanol; (B): 0.50 g or 1.00 mL of the acetone standard
without further purification. dissolved in 20.0 mL ethanol. After this mixing, a precipitate
was formed in each case, isolated through filtration, and dried
2.2. Instrumentation. For the chromatographic analysis, in vacuum [3, 24, 25].
Thermo Scientific Dionex Ultimate 3000 HPLC with a Acetone was added into its 2,4-DNPH derivatives by
ThermoAcclaim-C18 (15 cm × 4.6 mm × 3 𝜇m) column mixing 1.00 mL of a solution containing 200 mg/100 mL of
and UV-Vis DAD were used. The deionized water was 2,4-DNPH with 1.0 mL of H3 PO4 , and 4.00 mL of the human
18.2 MΩ⋅cm (Millipore Direct-Q3 UV) and was used serum. After 2 h, a 40.0 𝜇L aliquot was withdrawn and ana-
throughout the experiments. For the biochemical analyses, lyzed by the HPLC technique [3, 24–27]. Chromatographic
Journal of Analytical Methods in Chemistry 3
Table 1: Quantitative analysis data of acetone as its 2,4-dinitrophenylhydrazone derivative in human blood by HPLC.
Acetone levels by HPLC (mean value ± sd.)∗

Sample Subject Sex Age Blood glucose Urine ketone
Blood (mmol L−1 )∗∗
Group 1
Type 1 diabetes mellitus, Hashimoto
1 disease, systemic lupus F 21 197 ++ 15.00 ± 1.00
erythematosus
2 Diabetes mellitus (DM), pneumonia M 68 320 + 2.40 ± 0.20
3 DM, obesity, depression F 33 196 + 3.86 ± 0.10
4 Acute pancreatitis M 60 134 + 0.013 ± 0.001
5 Acute pancreatitis, obesity, F 33 117 + 0.22 ± 0.01
hyperlipidemia
6 DM F 37 175 + 0.24 ± 0.01
7 Gestational DM F 37 74 +++ 17.27 ± 1.00
Group 2
8 DM, hypothyroidemia M 80 340 − 0.42 ± 0.01
9 DM, hypertension F 82 248 − 3.20 ± 0.10
10 DM, hypertension, cerebrovascular F 77 202 − 3.10 ± 0.10
disease, acute kidney failure
11 Anemia, hypothyroidemia, M 52 110 − 0.033 ± 0.01
hyperlipidemia
12 DM, hypertension, hyperlipidemia F 55 137 − 1.53 ± 0.01
Hypertension, congestive heart
13 disease, chronic obstructive M 83 182 − 1.51 ± 0.01
pulmonary disease, cerebrovascular
disease
14 DM, larynx carcinoma, M 59 177 − 1.54 ± 0.01
hypothyroidemia
15 Hepatic failure, hypertension, M 87 111 − 2.13 ± 0.10
chronic kidney failure
∗
Number of repeated experiments, 𝑛 = 4, and ∗∗ reference value: 0.034–0.120 mmol L−1 with a mean value in plasma: 0.075 mmol L−1 (𝑛 = 20) [2].
separation was achieved in a ThermoAcclaim-C18 (15 cm × 2.6.1. Linearity. The linearity of the method was determined
4.6 mm × 3 𝜇m) column at UV-Vis DAD detector (𝜆 max at four concentration levels ranging from 0.5 to 20 mmol L−1 .
365 nm). The injection volume was 20.0 𝜇L and the detection The calibration curves were constructed by plotting the
was performed at 365 nm. The following gradient was used: peak area of the 2,4-DNPH derivative of acetone (𝑦) versus
(methanol/acetonitrile) (8 : 2) water 60 : 40 (v/v). Elution concentration of acetone (𝑥). The slope, 𝑦-intercept, and
was achieved at retention time (𝑡R ) 12.10 and flow-rate of correlation coefficient were calculated. To check the linearity,
1 mL min−1 . 𝐹 test also was applied [28–33].
2.5. Preparation of Calibration Standards. Standard cali- 2.6.2. Limit of Detection (LOD) and Limit of Quantification
bration curve was prepared with acetone (0.5, 2.5, 5.0, (LOQ). The LOD was estimated using signal-to-noise ratio of
10, and 20 mmol L−1 ) and 40 𝜇L 2,4-DNPH in acetoni- 3 : 1 or (3 s/m) and LOQ as 10 : 1 or (10 s/m), at which accuracy
trile. Human samples were prepared by adding 40 𝜇L 2,4- and standard deviation were within 20% as per ICH [28, 32,
DNPH and 500 𝜇L acetonitrile to 200 𝜇L human serum. 34, 35].
The calibration curve was constructed by plotting the
peak area of the 2,4-DNPH derivative of acetone (𝑦) ver- 2.6.3. Accuracy and Precision. Intraday accuracy and preci-
sus the concentration of acetone (𝑥, mmol L−1 ) by linear sion were performed for acetone at 5.0 mmol L−1 in replicate
regression (𝑛 = 4). The equation was found as 𝑦 = (𝑛 = 3). Interday accuracy and precision were achieved on
0.7361𝑥 + 0.0877 with a 0.9967, correlation coefficient three different days. Accuracy (expressed as recovery) and
(𝑅). precision (expressed as relative standard deviation) should
not deviate by ±15% of the nominal concentration [34].
2.6. Method Validation. The method proposed was validated 3. Results and Discussion
as described in ICH guidelines in parameters of linearity,
limit of detection and quantification, accuracy, and precision 3.1. The Chromatogram of 2,4-DNPH. Elution and recov-
[28]. ery problems were solved using HPLC with 2,4-DNPH as
Table 2: Intraday and interday precision and accuracy of the applied method.
Analyte Period of analysis Nominal concentration Mean concentration found Recovery% RSD% 𝑛
mmol L−1 mmol L−1
Acetone
Intraday 0.50 0.49 ± 0.01 98 2.04 3
Interday 0.50 0.48 ± 0.01 96 2.10 3
200 120
DNPH
Acetone
150 75
(mAU)
50
(mAU)
100
25
50
0 5 10 15 20 25
−20 (min)
0 5 10 15 20 25 Figure 2: : Typical HPLC chromatogram of 0.681 mmol L−1 acetone
(min) as its 2,4-DNPH derivative; for chromatographic conditions: see
Section 2.
Figure 1: The chromatogram of 2,4-DNPH; for chromatographic
conditions: see Section 2.
3500
derivatizing reagent. The chromatogram of 2,4-DNPH is

given in Figure 1. As can be seen from Figure 1, the retention 2000
(mAU)
time (𝑡R ) was obtained as 3.80 min.
3.2. Typical HPLC Chromatogram of Acetone as Its 2,4-DNPH 1000

Acetone
Derivative. The most efficient separation of acetone as its
2,4-DNPH was obtained with a ThermoAcclaim C18 column
(15 cm × 4.6 mm × 3 𝜇m) at retention time (𝑡R ) 12.10 min −500
and flowrate of 1 mL min−1 using a (methanol/acetonitrile) 0 2.5 5.0 7.5 10 12.5 15 17.5 20
water elution gradient. No elution problem for acetone as its (min)
2,4-dinitrophenylhydrazone derivative was observed. Typical
Figure 3: The HPLC chromatogram of 15 mmol L−1 acetone in
HPLC chromatogram of 0.681 mmol L−1 acetone as its 2,4- human blood from first patient of Group 1; for chromatographic
DNPH derivative is given in Figure 2. conditions: see Section 2.
3.3. Determination of Acetone in Human Blood by HPLC.

The HPLC chromatogram of 15 mmol L−1 acetone in human 20 mmol L−1 . The regression equation and correlation coef-
blood in the first patient of Group 1 is given in Figure 3. ficient were obtained as 𝑦 = 0.7361𝑥 + 0.0877 with a 0.9967,
correlation coefficient (𝑅).
3.4. Quantitative Determination of Acetone in Patients. In 𝐹 test applied to accuracy of calibration curve. For
our study, the patients were determined as high blood calibration curve, 𝐹critical value at 12 degrees of freedom (𝑝 =
glucose and urine ketone positive subjects (Group 1) and high 0.05) is 2.20. Experimental 𝐹 value is obtained as 1.26. So this
blood glucose and urine ketone negative example subjects value is smaller than 2.2, and calibration curve is linear. For
(Group 2). Test results were screened for hyperglycemia the human plasma samples, 𝐹critical value for calibration curve
and ketonuria. The blood glucose levels, given in Table 1, at 3 degrees of freedom (𝑝 = 0.05) is 3.18. Experimental value
vary between 110 and 320 mg/dL in our patients, except for is obtained as 2.03. So, this value is smaller than 𝐹critical value,
sample 7. Many patients in our study have DM disease, except and obtained values are appropriate.
samples numbered 4, 5, 11, 13, and 15 (Table 1).
3.5.2. Limit of Detection and Quantification. The LOD and
LOQ were found as 0.041 and 0.136 mmol L−1 , respectively.
3.5. Method Validation
3.5.1. Linearity. The calibration plot of peak area against 3.5.3. Accuracy and Precision. Accuracy and precision results
concentration was obtained linear in the range 0.5 to are summarized in Table 2. For intraday assay, the accuracy
O + −
− O
O H2 O H OH
R R1 R C R1 R C R1 R C R1
∙∙ +
NH2 + ∙∙
H2 O H NH
H NH NH
NH NH NH
NH H3 O+ /H2 O
NO2 NO2
NO2 NO2
NO2 NO2
NO2 NO2
+
H2 O H
+ ∙∙
H OH OH
R R1
C R C R1 R C R1 R C R1
N ∙∙ + NH NH
H2 O H N
∙∙
∙∙
NH −H3 O+ NH −H2 O NH −H2 O NH
NO2 NO2 NO2 NO2
NO2 NO2 NO2 NO2
Scheme 1: Mechanism of 2,4-dinitrophenylhydrazine to 2,4-dinitrophenylhydrazone.
for acetone in human plasma samples expressed as recovery interacts with the p-orbital of the carbonyl carbon resulting in
was found as 98%. For interday assay, the accuracy for acetone increased delocalization in the molecule. This stability would
in human plasma samples expressed as recovery was found as be lost by addition of a reagent to the carbonyl group. Hence,
96%. these compounds are more resistant to addition reactions.
Also with carboxylic acids there is the effect of the compound
3.6. Mechanism of 2,4-DNPH to 2,4-Dinitrophenylhydrazone acting as a base, leaving the resulting carboxylate negatively
charged and hence unable to be attacked by this nucleophile
Brady’s Test. 2,4-DNPH can be used to qualitatively detect [36, 37].
the carbonyl functionality of a ketone such as acetone or
aldehyde functional group. A positive test is signaled by a 4. Conclusions
yellow, orange, or red precipitate known as a dinitrophenyl-
hydrazone. If the carbonyl compound is aromatic, then the In this study, an analytical method was applied for the
precipitate will be red; if aliphatic, then the precipitate will quantitative determination of acetone in human blood. The
have a more yellow color [36]. The reaction between 2,4- determination was carried out using a UV-Vis DAD detector
DNPH and a ketone such as acetone is shown below: with HPLC. In most of our patients’ blood samples, high
level of acetone has been determined. Higher levels of acetone
RR󸀠 C = O + C6 H3 (NO2 )2 NHNH2
(1) have been measured in the patients who have high level of
󳨀→ C6 H3 (NO2 )2 NHNCRR󸀠 + H2 O blood glucose and positive urine ketone (Group 1). The HPLC
method based on the labeling of acetone with 2,4-DNPH
This reaction can be described as a condensation reaction, seems to offer a rapid, low cost, sensitive, selective, and
with two molecules joining together with loss of water. It reproducible methodology for quantification of the acetone
is also considered an addition-elimination reaction: nucle- level in clinical samples such as human blood. The HPLC
ophilic addition of the -NH2 group to the C=O carbonyl method described here overcomes many of the problems
group, followed by the removal of an H2 O molecule. The in the determination of acetone in biological fluids and
mechanism of 2,4-DNPH to 2,4-dinitrophenylhydrazone is the preanalytical errors. The volatile ketone is promptly
given in Scheme 1 [37]. stabilized by conversion into its DNPH derivative and rapidly
2,4-DNPH does not react with other carbonyl-containing determined without recourse to a solvent extraction step. The
functional groups such as carboxylic acids, amides, and method uses very inexpensive reagents. As can be stated by
esters. For carboxylic acids, amides, and esters, there is Brega et al., the proposed HPLC method can therefore be
resonance associated stability as a lone pair of electrons used to great advantage over current gas chromatographic
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Hindawi Publishing Corporation

Journal of Analytical Methods in Chemistry

Esin Akgul Kalkan,1 Mehtap Sahiner,2 Dilek Ulker Cakir,3

Correspondence should be addressed to Selehattin Yilmaz; [email protected]

Received 13 February 2016; Revised 26 April 2016; Accepted 4 May 2016

Academic Editor: Mohamed Abdel-Rehim

1. Introduction alcohol ingestions, drug toxicities, inborn errors of ketone

Acetone levels by HPLC (mean value ± sd.)∗

derivatizing reagent. The chromatogram of 2,4-DNPH is

time (𝑡R ) was obtained as 3.80 min.

3.2. Typical HPLC Chromatogram of Acetone as Its 2,4-DNPH 1000

3.3. Determination of Acetone in Human Blood by HPLC.

NO2 NO2 NO2 NO2

Scheme 1: Mechanism of 2,4-dinitrophenylhydrazine to 2,4-dinitrophenylhydrazone.

with ketoacidosis,” Clinica Chimica Acta, vol. 430, pp. 140–144,

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