Anal Bioanal Chem (2013) 405:1929–1935 DOI 10.1007/s00216-012-6658-3

ORIGINAL PAPER

Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides Sarojini J. K. A. Ubhayasekera & Johan Staaf & Anders Forslund & Peter Bergsten & Jonas Bergquist

Received: 4 September 2012 / Revised: 29 November 2012 / Accepted: 12 December 2012 / Published online: 11 January 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Circulating free fatty acids (FFAs) play important physiological roles as contributing components in cellular structure as well as energy utilization. Elevated levels of circulating FFAs are associated with metabolic aberrations in humans. FFAs differ in chain length and saturation and may be altered in metabolically dysregulated conditions, such as type 2 diabetes mellitus. Potentially, alterations in circulating levels of specific FFAs could also be important in terms of prognostic value. Various methods have been used to analyze FFAs. In this study, a straightforward and accurate method for the determination of FFAs in plasma has been established and evaluated, through conversion of plasma FFAs into acid fluorides followed by conversion to Weinreb amides (dimethylamide). The method is mild, efficient, selective, and quantitative for FFAs, when analyzed with capillary gas chromatography tandem mass spectrometry. Standard curves were linear over the range of 1,000–20,000 ng/mL with a correlation coefficient (r2) of 0.998, and coefficient of variation of triplicate analysis was <10 %. The gas chromatography–mass spectrometry (GC-MS)

Awarded an ABC Poster Prize on the occasion of the “21st Analysdagarna” held in Uppsala, Sweden from June 11–13, 2012. S. J. K. A. Ubhayasekera (*) : J. Bergquist Analytical Chemistry, Department of Chemistry—Biomedical Center and Science for Life Laboratory, Uppsala University, Box 599, 751 24 Uppsala, Sweden e-mail: [email protected] J. Staaf : P. Bergsten Department of Medical Cell Biology, Uppsala University, Box 571, 751 23 Uppsala, Sweden J. Staaf : A. Forslund Department of Women’s and Children’s Health, Uppsala University, 751 85 Uppsala, Sweden

technique was reproducible and repeatable, and recoveries were above 90 %. From the generated MS spectra, five specific FFAs were identified. An explicit interest was the quantification of palmitate (C16:0) and palmitoleate (C16:1), which have been connected with detrimental and positive effects on the insulinproducing beta cells, respectively. The results demonstrate the suitability of Weinreb amides for efficient and rapid isolation of FFAs in plasma, prior to quantitative GC-MS analysis. We suggest that the method can be used as a routine standardized way of quantifying FFAs. Keywords Free fatty acids . Obesity . Weinreb amides

Introduction Free fatty acids (FFAs) provide an essential source of energy for cells. In addition, different circulating FFAs also act in various manners, such as signaling molecules in cellular processes [1–3]. FFA levels differ between individuals, and elevated levels can be directly harmful for different types of cells [2, 4, 5]. Thus, to study the role of FFAs in humans, a method to accurately quantify different FFA species is needed. Most fatty acids (FAs) exist in the form of esters in lipids, and the FFAs comprise only a small part of total FAs. Therefore, prior to the analysis of FFAs of biological plasma samples, it is essential to remove the esterified FAs through extraction and separation procedures. Classically, lipids are extracted with solvent mixtures, as described by Folch et al. [6], Bligh and Dyer [7], and Hara and Radin [8]. The chromatographic separation techniques, such as thin-layer chromatography (TLC), liquid chromatography (LC), or solid-phase extraction (SPE), have been described for FFA separation from total FAs [9–11].

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However, these reported methods have several weaknesses, for instance being time consuming, oxidation of FAs, and poor recovery. In this context, additional methods have been used to analyze FFAs, including colorimetric, titration, and enzymatic approaches [12–14]. In routine practice, the latter methods are widely implemented, due to their simplicity and rapidity. However, the sensitivity of these methods is limited to total levels of FFAs in the plasma sample; hence, it is not possible to determine concentrations of individual FFA species. Therefore, LC and gas chromatography (GC) have been used to analyze the individual FFAs in different matrices [11, 15–17]. In recent years, capillary GC has been a vital analytical technique when combined with mass spectrometry (MS). This is a powerful technique for FA profiling with high sensitivity and reproducibility [15, 16]. The most important and critical issue for GC determination of FFAs is the sample preparation, which usually requires three different workup steps. The initial step is extraction of lipids, followed by enrichment of FFAs from other lipids, either by TLC or SPE, and finally the derivatization of FFAs into suitable derivatives prior to analysis. For the derivatization, methyl esters prepared at room temperature by alkali catalysts are commonly used instead of trimethylsilyl ether for FA profiling by capillary GC, equipped with a flame ionization detector (FID) with polar stationary phases [10, 15]. Despite several disadvantages in the preparation of methyl esters to achieve the low ionization potential, the carboxylic group needs to be derivatized with a reagent containing nitrogen for GC-MS analysis [15]. GC-MS separation of FA amides has been studied, mainly the picolinyl esters and 4,4dimethyl-2-oxazolines derivatives of FA [15]. However, for obtaining a sufficient derivatization yield, it takes a longer reaction time as well as a higher temperature [15]. To minimize the limitations, Tunoori et al. have introduced the Deoxo-Fluoro, which converts carboxylic acid to acid fluorides and then into Weinreb amides in the same flask at 0 °C [18]. Furthermore, Kangani et al. have developed this method with mild conditions together with a shorter sample preparation time [19]. In light of the potential importance of circulating FFA species, which may play different functions in the pathogenesis of diverse diseases, here, we report a simple and stable GC-MS method to quantify FFAs in plasma, using FFA amide derivatives (dimethylamide) with a fluorinating reagent (Deoxo-Fluoro) [18].

S.J.K.A. Ubhayasekera et al.

(C18:0), oleic acid (C18:1), and linoleic acid (C18:2), were obtained from Larodan Fine Chemicals AB (Malmö, Sweden). Dimethylamine and Deoxo-Fluoro reagent (bis(2-methoxyethyl)amino-sulfur trifluoride) were obtained from Sigma-Aldrich (Stockholm, Sweden). All other chemicals and solvents were of analytical grade and were obtained from Merck Euro lab AB (Stockholm, Sweden), unless otherwise stated. Study population The study population consisted of 50 morbidly obese children and adolescents, ages 3–18 years (Table 1). Samples were obtained from the pediatric obesity clinic at Uppsala University Children's Hospital. The study population is part of a larger cohort, referred to as Uppsala Longitudinal Study of Childhood Obesity. Informed consent was acquired, and the study has been approved by the local ethical committee, registration number 2010/036. Sample preparation Blood samples were collected following an overnight fasting period. The collection of blood was performed by venipuncture, using standard EDTA tubes (VACUETTE®, Greiner Bio-One). Plasma was obtained by centrifugation at 2,500×g for 10 min. Subsequently, plasma was aliquoted and stored at −70 °C until analysis. Lipid extraction A slightly modified method of lipid extraction by Dole et al. was used [20]. Sample analysis was done in duplicates. In brief, 200 μL of plasma and 12.8 μg of internal standard (C17:0) were thoroughly vortexed with 2 mL of organic solvent mixture (isopropanol/heptane/ 1 M hydrochloric acid (40:10:1, v/v/v)) for 30 min. Samples were then left at room temperature for 10 min. Water (2 mL) was added to the samples, and plasma lipid was extracted into 4 mL of heptane. During the extraction, the lipids were protected against

Table 1 Study population

Methods and materials Chemicals Standards of FAs, palmitic acid (C16:0), palmitoleic acid (C16:1), margaric acid (C17:0), stearic acid

Characteristics Number of subjects: Females Males Age ISO-BMI

50 28 22 11.5±0.5 >30 kg/m2

Free fatty acid determination in plasma by GC-MS

oxidation by the addition of 0.05 mg/mL butylated hydroxytoluene to the organic solvent mixture. The heptane was dried under nitrogen, and the dried lipids were dissolved in 200 μL of dichloromethane. Samples were stored at −20 °C until further derivatization. Derivatization of FFAs Diisoproylethylamine (10 μL) and dimethylamine (30 μL) were added to plasma lipid in 200 μL of dichloromethane. The mixture was placed on ice for 15 min, and 10 μL of Deoxo-Fluoro reagent was added to the samples. The samples were vortexed for a few seconds and kept on ice for 30 min. Subsequently, samples were kept at room temperature for 10 min. Water (2 mL) and heptane (4 mL) were added to the samples and mixed thoroughly. The upper layer of heptane was collected. The heptane layer was evaporated under nitrogen, and the dried amide derivative was redissolved in 200 μL of heptane prior to GC-MS analysis. Analytical thin-layer chromatography The derivatized plasma FFAs were analyzed by analytical precoated TLC plates (silica gel 60, 10 ×10 cm, 0.25 mm thickness; Merck, Eurolab AB, Stockholm, Sweden) to visually check the completeness of derivatization of plasma FFAs into corresponding amides. TLC references were standard FFAs (C16:0 and C16:1). The prepared TLC was developed in the solvent system hexane/diethyl ether/acetic acid (85:15:1, v/v/v). The TLC plate was quickly dried in atmospheric temperature and sprayed with 20 % phosphomolybdic acid solution (Sigma-Aldrich, Sweden). The plate was baked at 120 °C for 10 min, enabling color development. Identification of FFAs Analysis of FFAs in plasma was performed using a 8000 Top Series gas chromatograph and a AS800 auto-sampler (CE Instrument, ThermoQuest, Italia S.p.A., MI, Italy) coupled to a Voyager mass spectrometer with Xcalibur version 1.2 software (Finnigan, ThermoQuest, Manchester, UK). Separation of FFAs was performed on a nonpolar capillary column (DB-5MS, J&W Scientific, Folsom, CA, USA), 30 m×0.18 mm× 0.18 μm film thickness. Helium was the carrier gas at an inlet pressure of 80 kPa. The injector temperature was 250 °C, and the samples were injected using the splitless injection mode. The oven temperature was 130 °C for 1 min, then raised to 270 °C at a rate of 50 °C/min, and kept at this temperature for 10 min. Finally, the temperature was raised to 290 °C, at a rate of 1 °C/min for another 10 min. The mass spectra were

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recorded at electron energy of 70 eV, and the ion source temperature was 200 °C. The spectra were scanned in the range of 50–500m/z. Identification of the FFAs was done by comparing the mass spectra of standard samples of the FFAs (amides of authentic FAs) and their retention times. Chromatograms were collected in full scan mode (total ion current chromatogram, TIC). Quantification of the FFAs was accomplished using margaric acid (C17:0) as an internal standard due to its physical and chemical behavior similar to that of target analyte as well as its absence in plasma. The identification of FFA amides was determined using their retention times and m/z value of molecular ions generated from each FFA amide derivative. Quantification of FFA amides (area response) was accomplished with the help of the internal standard. Method validation The proposed method was validated using spiked plasma samples as described in the guidelines by the FDA (http:// www.fda.gov/cder/guidance/4252fnl.pdf.2001). Stock solutions of C16:0, C16:1, and internal standard (IS) were prepared independently at 1 mg/mL in ethanol and stored at −20 °C. Prior to the experiment, these solutions were serially diluted with the same solvent, and calibration and quality control solutions (QCs) were prepared. Linearity To assess the linearity, five different concentrations of palmitic acid (C16:0) and palmitoleic acid (C16:1) were separately mixed with a constant amount of internal standard (C17:0) over the concentration of 1–20 μg/mL. However, the nonspiked sample only with an IS was also analyzed for each analyte to confirm the absence of interferences. Samples were run in triplicates. Calibration curves were separately prepared by plotting the peak area ratio of each compound area of IS against the concentration of each compound. The linearity was determined by linear regression analysis. Precision and accuracy Quality control samples were used for precision and accuracy determination on intra-day and inter-day basis. The intra-day (n=3) and inter-day precisions (n=6) were determined for the spiked plasma samples. Limit of detection and quantification The detection limit (LOD) and the quantification limit (LOQ) were calculated using calibration curve of plasma

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spiked with C16:0 and C16:1. LOD was calculated using the formula 3.3 δ/S, and LOQ was obtained by the formula 10 δ/S; here, δ is the residual standard deviation of the regression line, and S is the slope of the regression line. Recovery Plasma samples were spiked in triplicates with known amounts of two different analytes at three different concentration levels (1, 5, and 10 μg/mL) and analyzed to determine recovery.

Results and discussion Obesity is an escalating worldwide problem among adults as well as children and adolescents [1, 21, 22]. The aims of the method development were to measure circulating levels of FFA in young obese patients and correlate the fatty acid levels with beta cell function and insulin resistance (unpublished data). Currently, there is a knowledge gap in understanding the involvement of FFAs in the early stages of obesity and type 2 diabetes mellitus in adolescents. Hence, the evaluation of FFA levels in biological samples is important. There is no standard method to analyze FFAs in routine analysis, since each laboratory has its own preferences. As a part of the study on lipids in juvenile obesity, our aim was to develop a gentle and efficient method that accurately quantifies FFAs in plasma. Therefore, we used one-pot synthesis that converts FFAs to Weinreb amides [23]. In this process, FFAs react with dimethylamine and bis(2-methoxyethyl amino-sulfur trifluoride (Deoxo-Fluoro)) at 0 °C in the presence of CH2Cl2 and N, N-diisopropylethylamine forming the fluoride derivative of FFA (acid fluoride) as shown in the reaction scheme (Fig. 1). In the formation of Weinred amide derivative of FFA, amine acts as a nucleophile to the intermediate acyl fluoride [18, 23, 24]. FFA amide derivatization is a precise method for the

Fig. 1 Reaction scheme of the conversion of FFA into Weinreb amide derivative of FFA (FFA = C16:1)

determination of FFAs due to its high volatility and stable chromatographic performance [19]. Precision is obtained because other FA species like FA esters and cholesterol esters, which exist in the tissue samples, are not affected by the presence of Deoxo-Fluoro reagent, which confers specificity to quantifying FFAs [19]. The completion of the derivatization of FFAs into FFA amides was frequently checked with TLC. There were no trace amounts of FFAs left in the sample after the completion of the reaction. Kangani et al. reported that this procedure gives >90 % pure yield of FFA amides [23]. Therefore, FFA amides were more suitable for quantifying FFAs. Quantification of FFA amides was performed using electron ionization GC-MS. Nitrogen moieties of amides proved to be of great importance in the MS analysis, which enabled the identification of the structure due to their simple fragmentation [18, 23]. The mass spectra obtained were simple and easy to interpret. The mass spectra of amide derivatives of FFAs exhibited strong molecular ions and fragmented peaks that can be used as diagnostic ions for elucidation of the structures of derivatives of FFA amides. In this study, we have presented mass spectra of the most common FFA amide derivatives in plasma (Fig. 2). Presently, it is not possible to compare the mass spectra from this study due to the unavailability of published data from other studies. The elution pattern of the GC separation was C16:1, C16:0, C17:0, C18:2, C18:1, and C18:0, where the molecular weights of the identified FFA amides are m/z 281, 283, 297, 307, 309, and 311, respectively (Fig. 2 and Fig. 3). The molecular characteristics such as length of the carbon chain, degree of the unsaturation, and the position of the first double bond determine the elution pattern [16]. With the GCMS setting used in this study, the total analytical time for the elution of the FFAs was 21 min (Fig. 3). We did confirm our findings based on the MS data. The results could not be compared to Kangani et al., who observed a similar elution pattern of FFA amides with a total chromatographic time of 23 min from the GCFID, but with poor baseline resolution [19]. Other

CH3(CH2)5CH=CH(CH2)7COOH + (CH3)2NH

N, N-diisopropylethylamine C H3OC2H5)2NSF3 (CH3)2NH 0˚C in CH2Cl2

[CH3(CH2)5CH=CH(CH2)7COF]

CH3(CH2)5CH=CH(CH2)7CON(CH3)2 + H2O

Free fatty acid determination in plasma by GC-MS

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a

d C16:1 (m/z 281)

C17:0 (m/z 297)

b

e C16:0 (m/z 283)

C18:1 (m/z 309)

f

c

C18:2 (m/z 307)

C18:0 (m/z 311)

Fig. 2 Full scan MS spectra of amide derivatives of FFA, (a) internal standard; (b) palmitic acid; (c) stearic acid; (d) palmitoleic acid; (e) oleic acid; (f) linoleic acid. Details of the chromatographic conditions are given in the experimental procedures

components in the total lipid extract elute after the FFAs (Fig. 3). Thus, it is vital to set the GC oven temperature to remove all other lipids from the column to reduce possible interference. The TIC was adopted for quantification of FFAs together with internal standard (C17:0). Peak areas were calculated by autointegral function of the instrumental work station. The described method was validated, and the linearity was determined by linear regression analysis. The correlation coefficients of linear regression (r2) of C16:0

and C16:1were 0.9980 and 0.9983, respectively. The quality of the linear fit was determined by percentage of coefficient of variation (CV%) of the data at each concentration that was tested in triplicates for both C16:0 and C16:1. The maximum CV% (4.7 %) for C16:1 was acceptable according to FDA recommendations. The LOD and LOQ were determined from the calibration curve of the plasma spiked with both analytes. The resulting LOD of C16:0 was 0.003 μg/mL and that of C16:1 was 0.004 μg/mL. Thus, the resulting

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LOQ were 0.010 and 0.013 μg/mL, respectively. The instrument precision was determined by multiple injections of the QC samples (Table 2), and CV% was less than 3.0 %. The intra-day and inter-day (6 days) precisions were determined at 0.05 μg/mL concentration tested in triplicates for both analytes. CV% of intraday precisions were 0.8 and 0.9 %, and inter-day precisions were 0.3 and 0.4 % for C16:0 and C16:1, respectively. The recovery percentages were above 90 %, and estimated CV% for both C16:0 and C16:1 was 1.4 and 2.6 %, respectively. The method was applied to assess the FFA profile in plasma samples obtained from juvenile obese individuals of different ages. The reason for selecting this study population was the expected variability in levels of FFAs between obese individuals. These individuals are at an increased risk of developing metabolic diseases [25]. Hence, it is important to investigate the levels of individual FFAs. A representative GC-MS chromatogram of FFAs isolated from one of the plasma samples is shown (Fig. 3). The aim was to accurately identify and quantify the most abundant FFAs, which turned out to be palmitic, palmitoleic, stearic, oleic, and linoleic acids (Table 3). The aim was successfully achieved and is expected to contribute to a better understanding of the role of FFAs in these individuals. We observed that the FFA profiles varied up to almost 20-fold between individuals (Table 3). The FFA levels found in the present study were generally higher compared to published values for adult individuals with type 2 diabetes mellitus [16, 19, 26]. Discrepancies between results may be due to differences in the study population,

Relative Abundance

Oleic

Palmitic Linoleic

Table 2 Instrument precision at the three different concentration levels Concentration (μg/mL)

Estimated concentration (n=3) Mean±SD (μg/mL)

CV (%)

C16:0

C16:1

C16:0

C16:1

0.05 1.0

0.06±2.37 1.10±4.33

0.05±4.37 1.32±8.77

3.12 1.43

5.10 2.63

3.0

2.04±15.91

2.43±12.29

4.54

4.27

sample preparation, and analytical method. An important methodological advancement reported in this study is the reduction in plasma sample size compared to the previous studies [16, 19, 26], which is of particular importance when examining pediatric populations. Also, whereas the study by Kangani et al. used SpeedVac for sample drying [19], the present study used nitrogen gas to avoid the oxidation and degradation of FFAs. The two most abundant FFAs in circulation are oleic and palmitic acid (Table 3). The saturated FFA palmitic acid has various negative effects on insulin-producing beta cells [2, 27]. In contrast, the unsaturated FFA palmitoleic acid and oleic acid have been connected with positive effects on beta cells and insulin sensitivity [27–29]. To what extent levels of palmitoleic acid affect insulin sensitivity and beta cell function in juvenile obese subject still needs to be determined. Further investigations concerning levels and effects of different FFAs in vivo are needed, where the method described in this paper is expected to be useful. The novel GCMS method is very practical in FFA profiling in routine analysis. It was developed to enable simultaneous determination of several FFAs in small plasma volumes. It is expected that the FFA measurements obtained by this method will provide information contributing to a greater understanding of obesity among children.

IS Stearic Palmitoleic

Table 3 FFA levels of study population FFA

Retention Time (min)

Fig. 3 A capillary column GC-MS chromatogram showing the separation of the common FFAs (amide derivatives) obtained from plasma samples. GC capillary column and chromatographic conditions are given in the methodology section. Peak identification (from left to right): palmitoleic acid, palmitic acid, internal standard, linoleic acid, oleic acid, stearic acid

Palmitoleic acid Palmitic acid Linoleic acid Oleic acid Stearic acid

1.25±0.10 (0.24–3.83) 5.46±0.19 (2.61–8.66) 2.41±0.16 (0.30–5.64) 8.96±0.43 (3.10–18.05) 2.17+0.12 (0.29–4.10)

Results are given as mean±SEM (in milligram per deciliter) and range

Free fatty acid determination in plasma by GC-MS Acknowledgments We would like to acknowledge Professor Paresh Dutta (Department of Food Science, Swedish Agricultural University, Uppsala, Sweden) for his help with the instrumentation. The Swedish Research Council (grants 621-2008-3562, 621-2011-4423 (J.B.) and 72X-14019 (P.B.)), EC FP7-project Beta-JUDO (grant 279153; P.B., J.B., and A.F.), and the Uppsala Regional Research Council (A.F. and P.B.) are gratefully acknowledged for financial support.

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