Elsevier

Microchemical Journal

Volume 132, May 2017, Pages 49-58
Microchemical Journal

Multiclass profiling of lipids of archaeological interest by ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-high resolution mass spectrometry

https://doi.org/10.1016/j.microc.2016.12.023Get rights and content

Highlights

  • Two UHPLC-APCI-TOFMS methods were developed for analysis of lipids in Iberian archaeological pottery.

  • Determination of TAGs and sterols is possible in just one chromatographic run.

  • Mass fragmentation pattern by APCI source is essential to determine TAG positional isomers.

  • TAG and sterol LOQs are below 5 ng g 1 in matrix.

  • Ruminant fat can be distinguished from non-ruminant fat by this methodology.

Abstract

In the present work, an analytical methodology for the comprehensive evaluation of multiclass lipids (including mono-, di- and triacylglycerols (MAGs, DAGs and TAGs) along with sterols) in archaeological ceramic vessels has been developed. It is based on the use of ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-high resolution mass spectrometry (UHPLC-APCI-HRMS) with a time-of-flight mass analyser operated in the positive ionization mode. The vaporizer temperature of the APCI source was found to exert a critical influence on the fragmentation exhibited by the different studied TAGs. This fragmentation information combined with accurate mass measurements enabled the unambiguous identification of the targeted species. From the chromatographic side, two different methods were developed using: (i) a 2.1 × 50 mm C18 high-throughput column (1.8 μm particle size), with MeOH/H2O 50:50 (v/v) and isopropanol (i-PrOH) as mobile phases and; (ii) a 4.6 × 150 mm C18 high-throughput column (with 1.8 μm particle size), with MeOH and i-PrOH as mobile phases. The first method involved a fast 12-min gradient, which enabled a fast and sensitive screening of the lipid content in the studied samples. The second 25-min confirmatory method provided a thorough TAGs separation. Both methods were successfully applied for the comprehensive analysis of lipid traces from archaeological ceramics with LOQs below 5 ng g 1 in most cases. The proposed methodology was applied on different ceramic samples, which belong to the Culture of the Iberians (6th–1st ct. BCE).

Introduction

The detection of organic residues preserved in archaeological samples provides key information, since it represents a source of evidence about the human activities over time. Insightful information have been extracted from the study of substances like resins [1], [2], alcoholic beverages [3], [4], [5], [6], waxes [7], [8], [9] and vegetal and animal fats [10], [11] in a wide variety of archaeological samples. The study of lipids remaining in pottery is interesting since the ceramics are the place where the persistence of residual organic matter is bigger either absorbed into the porous micro-structure of the clay wall or conserved in visible surface deposits [12]. Heron et al. demonstrated that other potential effects, such as contamination from soil components, or alterations by microbial activity, are minimal for lipids absorbed in potsherds [13]. Hence, the chemical analysis of archaeological ceramic samples is useful to determine the nature of the rests recovered, since it is possible to distinguish between their vegetable or animal origin. When the remains are established as animal origin, it may be even possible to distinguish whether the fats are from ruminant or non-ruminant animals [10], [14], [15]. Therefore, the examination of these compounds, together with other archaeological studies, can be helpful to define about feeding, ritual and domestic habits in the ancient human societies [8], [9], [11], [16], [17], [18], [19], [20], [21], [22].

Since the 1990s, different analytical methodologies based on mass spectrometry (MS) have been developed to explain the origin and composition of the remaining fats recovered from archaeological vessels. Among them, analytical methodologies employed for fatty acid profile studies such as high-temperature gas chromatography (HT-GC) or gas chromatography-mass spectrometry (GC–MS) provide the distribution of fatty acid methyl esters (FAMEs) and the positional isomers of monounsaturated fatty acids, thus confirming the (animal) origin of fatty content from the remains found in archaeological ceramic vessels [15], [23], [24]. The GC–MS methodology has been also used to distinguish the fat origin (animal or vegetal) and to detect the presence of other lipid classes such as sterols, n-alkanes, long-chain alcohols, wax esters, ketones, and mono-, di- and triacylglycerols [8], [16], [25], [26], [27]. Other variant of GC analysis is gas chromatography isotope-ratio mass spectrometry (GC-C-IRMS), which is based on the carbon isotope-ratio measurements for the determination of δ13C values of the major saturated fatty acids (palmitic and stearic acids) [14], [27], [28], [29]. The main drawback of GC methods is the prerequisite of an additional and time-consuming previous step in sample preparation, where derivatization or pyrolysis must be used, since the non-volatile analytes have to be transformed into suitable compounds amenable by GC–MS [14], [30], [31], [32], [33]. In order to avoid analyte derivatization, alternative methods based on high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) has been developed and applied to analyze specific lipid fractions such as triacylglycerols (TAGs) and sterols [34], [35].

The interest for TAGs studies in the archaeological field relies on their stability in the ceramic vessels and their essential inert properties [36]. Currently, the molecular speciation of TAGs is a complicated task due to the large variety of compounds that comprises its family and the positional isomers associated. To tackle this challenge, soft ionization techniques such as atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) hyphenated with triple quadrupole (TQ), quadrupole-time of flight (Q-TOF) or linear ion trap (LIT) mass spectrometers have been proposed to analyse TAGs isomers from archaeological ceramics [10], [15], [23], [24], [34], [36], [37], [38]. Due to the current increase in the number of studies which involve the analysis of lipid extracts from ceramic vessels, the development of methods for the rapid examination of a large number of archaeological samples in a short period of time, employing more time and resources only for positive samples is of great interest.

The aim of this work is to develop a new method for the study of TAGs and sterols from archaeological pottery, based on the use of ultrahigh-performance liquid chromatography atmospheric pressure chemical ionization-high resolution mass spectrometry (UHPLC-APCI-HRMS). The proposed approach consists of two steps: first a quick screening step using a short-UHPLC run (12 min) to obtain a rapid sample classification of the different lipids, followed by a thorough UHPLC separation (25 min). The latter run is just applied in the case of positive findings in order to provide a final confirmation of the targeted lipids. The method was applied to simultaneous determination of lipids of archaeological interest, covering TAGs and their transformation products, mono- and diacylglycerols (MAGs and DAGs, respectively), and steroid-type analytes as cholesterol and other sterols. To our knowledge, this is the first multiclass method for lipid profiling in archaeological ceramics covering different classes of compounds by UHPLC-MS. The proposed methodology has been satisfactorily applied to three ceramic samples from the Culture of the Iberians (6th–1st ct. BCE recovered in South and East-Northern Spain) [39], in which animal and vegetable fats were identified.

Section snippets

Chemical and reagents

All the solvents used were HPLC-grade. Chloroform (CHCl3), cyclohexane, isopropanol (i-PrOH) and methanol (MeOH) were obtained from Sigma-Aldrich (Madrid, Spain). A Milli-Q-Plus ultra-pure water system from Millipore (Milford, MA, USA) was used to obtain HPLC-grade water. Formic acid was purchased from Fluka (Buchs, Switzerland). As derivative reagents in GC analysis, N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, ≥ 99%) and chlorotrimethylsilane (TMCS, ≥ 99.0%) were obtained from

Ionization source and MS parameters

A MS method was developed to allow the simultaneous determination of different lipids of archaeological interest, covering TAGs, and their transformation products (MAGs and DAGs), and also steroid-type species. Previous studies of these compounds conducted by LC–MS analysis have shown that both APCI and ESI led to acceptable results to detect TAGs and sterols [35], [38], [40], [41]. In our study, the APCI source was selected as both TAGs and sterols could be detected in the same chromatographic

Conclusions

In this work, two chromatographic methods based on UHPLC hyphenated to high-resolution mass spectrometry have been reported for multiclass determination of lipids in archeological pottery. To our knowledge, these are the first multiclass methods for lipid profiling covering different classes of compounds as acylglycerols and sterols in this kind of samples by UHPLC-HRMS. Method 1 can be used for quick sample screening, allowing to focus the efforts only in positive samples, which are

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by funds from the University Research Institute for Iberian Archaeology (University of Jaén, Spain), by Ministry of Economy and Competitiveness through projects HAR2011-22994 and CTQ-2012-34297, partially co-financed with FEDER funds. We also acknowledge support from Plan Propio of Universidad de Jaén (Ref. 2016/CL042 (T2-I)).

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