Development of gas chromatography–mass spectrometry following headspace single-drop microextraction and simultaneous derivatization for fast determination of the diabetes biomarker, acetone in human blood samples

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Abstract

Acetone in human blood has been regarded as the diabetic biomarker. Acetone analysis is used as the accessorial diagnosis tool for diabetes. In this work, a simple, solvent-free and low-cost technique was developed for fast determination of acetone in blood samples, which was based on headspace single-drop microextraction and simultaneous derivatization followed by gas chromatography–mass spectrometry (GC–MS). Acetone in blood was headspace extracted by using the microdrop solvent containing a derivatization agent of O-(2,3,4,5-pentafluorobenzyl)hydroxylamine (PFBHA). The extracted acetone reacted with PFBHA in the microdrop, and rapidly formed acetone oxime. Finally, the derivative in the microdrop was detected by GC–MS. The parameters of headspace single-drop microextraction and simultaneous derivatization were studied, and the method validations were also studied. The proposed method was tested by the application to the determination of acetone in blood samples from controls and diabetic patients. The results show that headspace single-drop microextraction and simultaneous derivatization followed GC–MS is a simple, rapid, solvent-free and sensitive method for the determination of acetone in blood, and also a potential tool for diagnosis of diabetes.

Introduction

In mammals, acetone is derived from decarboxylation of acetoacetate and the dehydrogenation of isopropanol [1]. Acetoacetate is the major source of acetone in mammals, which is generated by dextrose metabolism and lipolysis. Due to that dextrose metabolism and lipolysis can be increased by relative or absolute lack of insulin, acetone concentration can elevate by at least two degrees of magnitude in the blood of diabetic patients [2], [3]. Even in the treated diabetic patients, its concentration is much higher than that in normal population [4]. In addition, the defect of complex enzyme of propionyl-CoA carboxylase (EC 6.4.1.3) or the methylmalonyl-CoA mutase (EC 5.4.99.2) also leads to the increase of acetone concentration [5], [6]. Acetone has been regarded as the biomarker of diabetes and other diseases [7], [8]. Recently, Kalapos pointed out that acetone analysis could be used as the accessorial diagnosis tool for diabetes [1].

Chromatographic technique is the ideal tool for quantification of the disease biomarker of acetone in biological fluids. Brega and coworkers developed liquid chromatography (LC) for analyzing acetone in plasma samples after derivatization with 2,4-dinitrophenyl-hydrazine (DNPH) [9]. Gas chromatography (GC) combined with sample enrichment techniques such as solid sorbents, evacuated stainless canisters and solid-phase microextraction (SPME) was also developed for direct determination of acetone in breath and plasma [10], [11], [12], [13], [14], [15]. Considering its nature of high activity and volatility, derivatization of acetone was required prior to GC analysis. Recently, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed as the derivatization agent of carbonyls, and applied to the determination of acetone and other carbonyls in biological samples and other samples [16], [17], [18]. A simple, rapid and solvent-free technique, solid-phase microextraction (SPME) with in-fiber derivatization was developed for the analysis of aldehydes in complex matrices [19], [20], [21], [22]. However, these SPME methods still have the following shortcomings: (1) SPME fiber is relatively expensive; (2) the SPME polymer coating is fragile and easily broken; (3) sample carryover is sometimes difficult or impossible to be eliminated.

Recently, a fast, simple, inexpensive, and virtually solvent-free sample preparation method has been devised for the extraction of analytes from water; this technique is known as liquid-phase microextraction (LPME) or single drop microextraction (SDME) [23], [24], [25], [26]. SDME combined extraction, concentration, and sample introduction in a single step. It has been used for the extraction of dialkylphthalates, nitroaromatics, polycyclic aromatic hydrocarbons, organochlorine compounds, triazine herbicides, cocaine, and endosulfans [27], [28], [29], [30], [31], [32]. To analyze volatile compounds in dirty samples, in 2001, Theis et al. [33] introduced headspace single-drop microextraction (HS-SDME), where a microdrop of organic solvent was suspended in the headspace of the analyte solution and used for headspace extraction and concentration of the volatile analytes in the solution. HS-SDME technique was widely applied to environmental and biomedical analysis [34], [35], [36], [37], [38], [39]. In our previous study [40], HS-SDME was applied to the analysis of aldehydes in human blood after derivatization with PFBHA.

In this work, gas chromatography–mass spectrometry (GC–MS) following SDME with simultaneous derivatization was developed for fast determination of the diabetes biomarker, acetone in human blood. Extraction parameters were studied, and method validations were also investigated.

Section snippets

Chemicals and blood samples

Acetone and butanone (internal standard) was purchased from Sigma (St. Louis, MO, USA). Acetone stock standard with the concentration level of 10 mM was prepared in methanol, and was stored in a freezer at −4 °C. The internal standard solution (10 mM) was prepared by dissolving butanone in methanol. O-(2,3,4,5-pentafluorobenzyl)hydroxylamine hydrochloride (98%, PFBHA) was purchased from Sigma (St. Louis, MO, USA). Decane was purchased from Chemical Agent Company, Shanghai, China. A 20 mg mL−1 PFBHA

Results and discussion

The aim in this work was to develop and optimize HS-SDME with simultaneous derivatization for fast determination of the diabetic biomarker of acetone in blood. In our previous studies [35], [36], it has been shown that the reaction of carbonyls with PFBHA is very rapid and complete, and the formed oxime is very compatible with GC or GC–MS analysis. In this method, acetone in blood was headspace extracted and concentrated by the microdrop solvent, and then fast reacted with PFBHA and formed

Conclusions

In this work, headspace single-drop microextraction and simultaneous derivatization has successfully been developed for fast determination of the diabetic biomarker of acetone in blood. Due to extraction, concentration and derivatization in single step, headspace single-drop microextraction and simultaneous derivatization technique provided a simple, rapid and sensitive approach to quantification of acetone in blood. Moreover, 2 μL solvent was required in each analysis, so, this method is very

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