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
Acetone is the simplest ketone compound. In general, acetone is not considered harmful, and the World Health Organization has not classified acetone as carcinogenic. However, its prolonged inhalation can not only cause irritation of the mucous membranes, headaches, confusion, and narcotic effects, but lead to coma as well [
For etiological reasons, acetonaemia is classified to be of endogenous and exogenous origin [
A lot of medical, chemical, and medicolegal investigations have been carried out with the determination of acetone in blood and other biological fluids [
In this paper, we present a rapid and simple HPLC technique using 2,4-DNPH as a derivatizing reagent for quantitative determination and metabolomic research of acetone in biological fluid such as human blood.
2,4-DNPH (Sigma-Aldrich, 97%), acetone (Merck, 99.8%), acetonitrile (Sigma-Aldrich, 99.8%), and methanol (Merck, 99.8%) were used in this study. All other chemicals were of analytical reagent grade and used without further purification.
For the chromatographic analysis, Thermo Scientific Dionex Ultimate 3000 HPLC with a ThermoAcclaim-C18 (15 cm
In the first stage of our study, following a 12-hour fasting venous blood samples were taken from patients admitted to hospital of the Faculty of Medicine, Canakkale Onsekiz Mart University. The human blood and urine samples were directly collected into a tube (without a collection device) between 08:30 and 11:00 am. Clinical Biochemistry Laboratory blood and urine glucose tests were studied for routine biochemistry using a urine autoanalyzer. Test results were screened for hyperglycemia and ketonuria. The patients were divided into high blood glucose and urine ketone positive subjects (Group 1) and high blood glucose and urine ketone negative example subjects (Group 2). The patients with hyperglycemia were 8 females and 7 males (age: 21–87;
Quantitative analysis data of acetone as its 2,4-dinitrophenylhydrazone derivative in human blood by HPLC.
Sample | Subject | Sex | Age | Blood glucose | Urine ketone | Acetone levels by HPLC (mean value ± sd.) |
---|---|---|---|---|---|---|
Blood (mmol L−1) |
||||||
|
||||||
1 | Type 1 diabetes mellitus, Hashimoto disease, systemic lupus erythematosus | F | 21 | 197 | ++ | 15.00 ± 1.00 |
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, hyperlipidemia | F | 33 | 117 | + | 0.22 ± 0.01 |
6 | DM | F | 37 | 175 | + | 0.24 ± 0.01 |
7 | Gestational DM | F | 37 | 74 | +++ | 17.27 ± 1.00 |
|
||||||
|
||||||
8 | DM, hypothyroidemia | M | 80 | 340 | − | 0.42 ± 0.01 |
9 | DM, hypertension | F | 82 | 248 | − | 3.20 ± 0.10 |
10 | DM, hypertension, cerebrovascular disease, acute kidney failure | F | 77 | 202 | − | 3.10 ± 0.10 |
11 | Anemia, hypothyroidemia, hyperlipidemia | M | 52 | 110 | − | 0.033 ± 0.01 |
12 | DM, hypertension, hyperlipidemia | F | 55 | 137 | − | 1.53 ± 0.01 |
13 | Hypertension, congestive heart disease, chronic obstructive pulmonary disease, cerebrovascular disease | M | 83 | 182 | − | 1.51 ± 0.01 |
14 | DM, larynx carcinoma, hypothyroidemia | M | 59 | 177 | − | 1.54 ± 0.01 |
15 | Hepatic failure, hypertension, chronic kidney failure | M | 87 | 111 | − | 2.13 ± 0.10 |
In the second stage of the study, to determine the probable positive acetone, its quantitative analysis was carried out in biological fluids using the HPLC technique. The blood samples were immediately prepared for HPLC analysis carried out within 8 hours after sample collection. A method for the determination of acetone in human blood by HPLC was developed. Plasma specimens were deproteinized with acetonitrile (1 : 1, v/v); 2,4-DNPH is added to the supernatant (filtered blood samples) and treated with acetonitrile (2 : 1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using ThermoAcclaim-C18 (15 cm
The quantitative analysis of acetone using HPLC was performed after labeling with 2,4-DNPH. The 2,4-dinitrophenylhydrazone standards were prepared by mixing A and B solutions: (A): 0.40 g of 2,4-DNPH dissolved in 2.00 mL of H2SO4 + 3.00 mL of H2O + 10.0 mL ethanol; (B): 0.50 g or 1.00 mL of the acetone standard dissolved in 20.0 mL ethanol. After this mixing, a precipitate was formed in each case, isolated through filtration, and dried in vacuum [
Acetone was added into its 2,4-DNPH derivatives by mixing 1.00 mL of a solution containing 200 mg/100 mL of 2,4-DNPH with 1.0 mL of H3PO4, and 4.00 mL of the human serum. After 2 h, a 40.0
Standard calibration curve was prepared with acetone (0.5, 2.5, 5.0, 10, and 20 mmol L−1) and 40
The method proposed was validated as described in ICH guidelines in parameters of linearity, limit of detection and quantification, accuracy, and precision [
The linearity of the method was determined at four concentration levels ranging from 0.5 to 20 mmol L−1. The calibration curves were constructed by plotting the peak area of the 2,4-DNPH derivative of acetone (
The LOD was estimated using signal-to-noise ratio of 3 : 1 or (3 s/m) and LOQ as 10 : 1 or (10 s/m), at which accuracy and standard deviation were within 20% as per ICH [
Intraday accuracy and precision were performed for acetone at 5.0 mmol L−1 in replicate (
Elution and recovery problems were solved using HPLC with 2,4-DNPH as derivatizing reagent. The chromatogram of 2,4-DNPH is given in Figure
The chromatogram of 2,4-DNPH; for chromatographic conditions: see Section
The most efficient separation of acetone as its 2,4-DNPH was obtained with a ThermoAcclaim C18 column (15 cm
: Typical HPLC chromatogram of 0.681 mmol L−1 acetone as its 2,4-DNPH derivative; for chromatographic conditions: see Section
The HPLC chromatogram of 15 mmol L−1 acetone in human blood in the first patient of Group 1 is given in Figure
The HPLC chromatogram of 15 mmol L−1 acetone in human blood from first patient of Group 1; for chromatographic conditions: see Section
In our study, the patients were determined as high blood glucose and urine ketone positive subjects (Group 1) and high blood glucose and urine ketone negative example subjects (Group 2). Test results were screened for hyperglycemia and ketonuria. The blood glucose levels, given in Table
The calibration plot of peak area against concentration was obtained linear in the range 0.5 to 20 mmol L−1. The regression equation and correlation coefficient were obtained as
The LOD and LOQ were found as 0.041 and 0.136 mmol L−1, respectively.
Accuracy and precision results are summarized in Table
Intraday and interday precision and accuracy of the applied method.
Analyte | Period of analysis | Nominal concentration |
Mean concentration found |
Recovery% | RSD% |
|
---|---|---|---|---|---|---|
Acetone | Intraday | 0.50 | 0.49 ± 0.01 | 98 | 2.04 | 3 |
Interday | 0.50 | 0.48 ± 0.01 | 96 | 2.10 | 3 |
Mechanism of 2,4-dinitrophenylhydrazine to 2,4-dinitrophenylhydrazone.
2,4-DNPH does not react with other carbonyl-containing functional groups such as carboxylic acids, amides, and esters. For carboxylic acids, amides, and esters, there is resonance associated stability as a lone pair of electrons interacts with the p-orbital of the carbonyl carbon resulting in increased delocalization in the molecule. This stability would be lost by addition of a reagent to the carbonyl group. Hence, these compounds are more resistant to addition reactions. Also with carboxylic acids there is the effect of the compound acting as a base, leaving the resulting carboxylate negatively charged and hence unable to be attacked by this nucleophile [
In this study, an analytical method was applied for the quantitative determination of acetone in human blood. The determination was carried out using a UV-Vis DAD detector with HPLC. In most of our patients’ blood samples, high level of acetone has been determined. Higher levels of acetone have been measured in the patients who have high level of blood glucose and positive urine ketone (Group 1). The HPLC method based on the labeling of acetone with 2,4-DNPH seems to offer a rapid, low cost, sensitive, selective, and reproducible methodology for quantification of the acetone level in clinical samples such as human blood. The HPLC method described here overcomes many of the problems in the determination of acetone in biological fluids and the preanalytical errors. The volatile ketone is promptly stabilized by conversion into its DNPH derivative and rapidly determined without recourse to a solvent extraction step. The method uses very inexpensive reagents. As can be stated by Brega et al., the proposed HPLC method can therefore be used to great advantage over current gas chromatographic methods; it can be used in experiments requiring multiple samples and specific activity determination for the routine measurement of acetone in diabetic patients and in biological monitoring of exposed workers. The use of very small sample amounts makes this a favourable method in pediatrics [
We also presented acetone as a useful tool for the HPLC-based metabolomics investigation of endogenous metabolism and quantitative clinical diagnostic analysis. In the medical and medicolegal practice, the level of acetone could not be characteristic for a specific disease. However, the detection and early identification of acetone could be used as an initial indicator of detection of all these physiopathological conditions [
The authors declare no conflict of financial, academic, commercial, political, or personal interests.
This study was supported by Canakkale Onsekiz Mart University Scientific Research Projects Commission, Canakkale/Turkey (Project no. TSA 2015-497), and was performed in Nanoscience and Technology Research and Application Center (NANORAC). The authors would like to thank all participants who kindly provided human blood and urine samples for the study and those who helped in the collection of samples at Canakkale Onsekiz Mart University Application and Research Hospital.