**3. Results**

## *3.1. Biochemical Analysis*

Assessment of ketone bodies is an important practice more especially in clinical institutions to check and monitor for diabetic ketoacidosis (DKA) and to ascertain whether breath acetone can be used in monitoring and controlling the disease. The high amount of ketone bodies in the blood is usually an indicator that the catabolism of fatty acid is greater than the one for carbohydrates [29,30]. In this study, 30 diabetic and 28 non-diabetic patients' fasting serums were analyzed for ketone bodies (acetoacetate and beta-hydroxybutyrate) using the Abcam® acetoacetate and beta-hydroxybutyrate assay kits (Figure 3). Other clinical data, which include blood glucose, glycated hemoglobin, total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein were also measured to confirm the state of diabetes mellitus (Table 1).

**Figure 3.** Scatter plot for plasma blood glucose, acetoacetate, beta-hydroxybutyrate and breath acetone in (**a**) type 2 diabetic and (**b**) non-diabetic mellitus patients.


**Table 1.** Clinical data of type 2 diabetes mellitus (DM) and non-diabetes mellitus.

Data in mean ± standard deviation (SD), BMI (body mass index), HDL (high-density lipoprotein) and LDL (low-density lipoprotein).

Breath acetone from both diabetic and non-diabetic patients was analyzed with the HS-SPME/GC-MS. As expected, guided by literature, a high amount of breath acetone was observed in diabetic patients (more than 0.8 ppm), as opposed to their non-diabetic (less than 0.8 ppm) counterparts. The mean values of acetoacetate in diabetic and non-diabetic patients were 0.09 mmol/L and 0.05 mmol/L, respectively. The mean β-hydroxybutyrate was also higher in diabetes patients, 0.46 mmol/L), as compared to the non-diabetes patients 0.25 mmol/L. As shown in Table 1, plasma glucose and total cholesterol were slightly higher in type 2 diabetes mellitus than non-diabetes mellitus. Triglycerides, HDL cholesterol, LDL cholesterol, β-hydroxybutyrate and acetoacetate levels were not significantly di fferent between the two groups.

#### *3.2. Breath Acetone Analysis Using HS-SPME*/*GC-MS*

In this study, HS-SPME/GC-MS analysis was applied to determine the concentration of acetone in the breath of 30 diabetic and 28 non-diabetic mellitus patients. Given the small mass of acetone (58 amu) and its volatility, the acetone in breath was first derivatized with a derivatizing reagent, pentafluorobenzyl-hydroxylamine-hydrochloride (PFBHA) prior to the GC-MS analysis. The reaction of acetone in the breath with a derivatizing agen<sup>t</sup> (PFBHA) forms a very stable acetone-oxime, and Figure 4 shows the mass spectrum of the acetone oxime with a base peak at *m*/*z* 181 which was extracted for quantitative purposes. The amount of oxime formed on the fiber is proportional to the acetone concentration in the breath. Acetone concentration higher than 1.8 ppm was observed in some of the diabetic breath (Figure 3a) and acetone concentration lower than 0.8 ppm was observed (Figure 3b). The GC spectrum of both diabetic and non-diabetic mellitus volunteers are available on the supplementarary results in Figure S1.

**Figure 4.** The gas chromatography-mass spectrometry (GC-MS) mass spectrum of acetone-oxime.

Determination of acetone in breath using HS-SPME/GC-MS with on-fiber derivatization yielded satisfactory precision and an excellent sensitivity with a simple procedure. The present method is a potential tool for a non-invasive diagnosis and monitoring of diabetes mellitus and ketoacidosis.

#### *3.3. Correlation Studies of Breath Acetone and Blood Ketone Bodies*

 

We observed the correlation coe fficient of*r* = 0.897 between breath acetone and plasma acetoacetate, and we further observed a good correlation of *r* = 0.821 between breath acetone and plasma beta-hydroxybutyrate. This shows a positive indicator of using acetone as a non-invasive biomarker of diabetes mellitus. The blood glucose and acetone also demonstrated a good correlation. The results are found in the Supplementary Material, Figure S2.
