Elsevier

Journal of Chromatography A

Volume 1216, Issue 9, 27 February 2009, Pages 1450-1457
Journal of Chromatography A

Analysis of fatty acids in sputum from patients with pulmonary tuberculosis using gas chromatography–mass spectrometry preceded by solid-phase microextraction and post-derivatization on the fiber

https://doi.org/10.1016/j.chroma.2008.12.039Get rights and content

Abstract

A method based on solid-phase microextraction (SPME) and post-derivatization on the fiber coupled to gas chromatography–mass spectrometry (GC–MS) was developed for the analysis of fatty acids in sputum from patients with pulmonary tuberculosis. The sputum specimens were digested, hydrolyzed, extracted, derivertized, injected and analyzed without cultivation or isolation of the microorganism. Under optimized conditions, the relative standard deviations (RSD, n = 5) for all analytes were below 17% and the limits of detection varied from 1.68 (C24:0) to 150.4 μg L−1 (C12:0). Good linearity was observed for all the fatty acids studied except for C12:0 within a wide concentration range of three orders of magnitudes with the correlation coefficients ranging from 0.91 (C24:0) to 0.99 (C14:0). Fatty acids in sputum specimens from 21 persons were directly analyzed using the proposed method. The results show that in all the sputum specimens from patients, who were clinically diagnosed with tuberculosis (TB), tuberculosis stearic acid (TBSA) was detected, while in all the sputum samples from persons without TB, TBSA was not found. The possibility of using the proposed method to detect mycobacterium tuberculosis (MTB) via the identification of TBSA in sputum was discussed. The comparison with other methods including sputum culture and microscopy of direct smears indicated that the proposed method is fast and sensitive for the analysis of fatty acids in sputum and offers an alternative for the detection of MTB in sputum.

Introduction

Tuberculosis (TB) is an ancient infectious disease caused by respiratory infection from the gram positive bacteria Mycobacterium tuberculosis (MTB). In recent years, TB has re-emerged as a major world health problem [1], [2], [3]. It is reported that approximately one-third of the world's population is infected with MTB, and almost nine million new cases of TB and approximately two million TB deaths occur annually [4], [5], [6]. Hence, TB remains a major world health problem and a method for the rapid screening of TB is still needed.

Many methods have been developed for the diagnosis of TB [7], [8]. Sputum culture is still the reference method for the diagnosis of pulmonary TB, but this method is too time-consuming because the organism is slow-growing. Although TB rapid cultivation detection technique is in clinical use, the fluorescent reagent which was used in this method is expensive and easily decomposed [9], [10]. Microscopy of direct smears for MTB has been recommended by World Health Organization for developing countries and is now the most commonly used method for diagnosis of TB. A major disadvantage of this method is its low sensitivity since the number of MTB in sputum and other clinical specimens is often very low. Though efforts have been made to concentrate the sputum samples, the sensitivity of this method is still not satisfactory [10], [11], [12], [13]. Polymerase chain reaction (PCR) method seems most promising among the newly developed methods for rapid diagnosis of TB, but this method often has a large amount of false positive results [14], [15]. Therefore, the development of a new inexpensive, sensitive and specific method for rapid diagnosis of TB is urgently needed.

During the last decades, detection of bacterial biomarkers using gas chromatography–mass spectrometry (GC–MS) has become a widely accepted and rather promising technique in clinical microbiology [16], [17], [18], [19]. These analytical methods have an advantage over traditional methods because they do not require the isolation of pure cultures for the identification of microorganisms owing to their high resolution. In addition, the high sensitivity of the methods makes it possible to detect bacteria without preculture and to shorten the time needed for a positive identification of bacteria greatly. Tuberculosis stearic acid (TBSA) is a characteristic component of the cellular fatty acids of mycobacteria and it has been shown that TBSA can serve as a marker in the detection of MTB in clinical specimens [20], [21], [22], [23], [24], [25]. As a powerful analytical method, GC–MS has also been used to detect TBSA in order to develop a method for the rapid diagnosis of TB [21], [22], [23]. However, the number of MTB in sputum and other clinical specimens is often very low, and thus the content of TBSA is very low. In addition, the complex matrix of the clinical specimens makes it difficult to be analyzed directly. To our knowledge, most of the work that has been done used solvent extraction as a sample preparation method [20], [21], [22], [24]. Stir bar sorptive extraction was also reported by Stopforth et al. [23] as a sample pretreatment in the detection of TBSA. It is well known that the solvent extraction method shows many disadvantages [26]. Firstly, it requires large volumes of organic solvents. Secondly, the extracts have to be concentrated prior to analysis.

Solid-phase microextraction (SPME) is an attractive sample preparation technique, which is inexpensive, time efficient, and solvent-free. It requires minimal sample treatment and simple instrumentation and integrates the extraction, preconcentration and sample introduction in a single process [27]. Owing to the advantages above, it has been widely and successfully used in the determination of volatile and semi-volatile compounds in various matrices by coupling to GC or other analytical approaches [28], [29].

Despite of its numerous advantages and comprehensive applications, SPME also presents some drawbacks. Particularly, the low sensitivity attainable for polar and nonvolatile analytes, such as medium and long chain fatty acids, is generally recognized as an important limitation of the SPME technique [30]. Derivatization is an important alternative that has been explored in order to enhance the sensitivity of SPME for these analytes. In addition, because of the high polarity or low volatility of medium and long chain fatty acids, which often leads to poor sensitivity and reproducibility of GC, derivatization is also needed to obtain satisfactory results. The utility of coupling derivatization to SPME has been extensively documented for a wide variety of matrices and analytes [31], [32], [33]. We have coupled in situ esterification and headspace SPME to GC–MS for the analysis of fatty acids in lung tissues using a homemade SPME fiber [34]. This method is appropriate for the dry samples since the esterification reaction is sensitive to water. Coupling direct immersion SPME (DI-SPME) and on-fiber post-derivatization to GC can give satisfactory results for the analysis of polar and nonvolatile analytes in aqueous mediums [35]. But to date, we are not aware of any report on the analysis of fatty acids in a complex matrix like sputum by coupling DI-SPME and post-derivatization on the fiber to GC–MS.

Here, a method for the analysis of fatty acids in sputum from patients with pulmonary tuberculosis was developed by DI-SPME and post-derivatization on the fiber before GC–MS. The SPME conditions as well as the derivatization conditions were optimized. In order to minimize the matrix effect, a fatty acid-free sputum blank was used for the method validation. Fatty acids in the sputum specimens from persons without TB as well as those who were clinically diagnosed with TB were analyzed using the proposed method. Finally, the results were compared with those obtained by using the smear method and the traditional cultivation method.

Section snippets

Instrumentation

A homemade SPME syringe with a sol–gel derived butyl methacrylate/hydroxy-terminated silicone oil (BMA/OH-TSO) fiber [36] was used to extract fatty acids. A model DF-1 magnetic stirrer (Jintan, China) was employed for temperature control and stirring during extraction and derivatization. GC–MS spectra were recorded on a Hewlett-Packard 6890N gas chromatograph coupled with a Hewlett-Packard 5973N mass spectrometer equipped with a MSD chemstation software and a HP-1 fused silica capillary column

Optimization of DI-SPME process

The influence of temperature on the extraction of fatty acids was shown in Fig. 1. It can be seen that the extraction yields of all fatty acids have an increase with the increase of temperature. 80 °C was set in the following experiments for DI-SPME of fatty acids.

Fig. 2 represents the extraction time profile for fatty acids. It was shown that 1.5 h is sufficient for C17:0, C18:0 and C20:0 to achieve the highest extraction yields. In the case of C12:0, C14:0, C16:0, C19:0 and C24:0, an

Conclusion

A method for the analysis of fatty acids in sputum from patients with pulmonary tuberculosis was developed by GC–MS preceded by DI-SPME and post-derivatization on a homemade BMA/OH-TSO fiber derived using sol–gel technique and was successfully applied to direct detection of MTB in sputum without preculture or isolation of the microorganism via the characterization of TBSA. The method is sensitive, precise, accurate and has a wide linear range. The comparisons with the smear method and the

Acknowledgement

This work was kindly supported by the National Natural Science Foundation of China (grant no. 20375028).

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