An efficient separation and quantification of the individual neutral and polar lipid classes and their constituent fatty acids was achieved by the combination of two different detection techniques: Iatroscan TLC-FID and GC-FID. The solvent composition and ratio of development system, the sample size, the fidelity, and precision were tested in order to estimate the effectiveness of separation of individual neutral and polar lipid classes and the quantitative reproducibility of the Iatroscan TLC-FID technique. GC-FID method, with a high-quality capillary column, allowed sensitive and reproducible fatty acid qualitative and quantitative analyses, separation of fatty acid structural isomers (e.g., n-C16:0, iso-C16:0 and anteiso-C16:0), positional isomers (e.g., C18:1
Fat and fatty acids especially polyunsaturated ones contribute to important aspects of fish, meat, and plant products’ quality and are critical for their nutritional and sensory value. Therefore, it is required to improve methods for the separation of total lipids into their neutral and polar fractions and for the analysis of their fatty acids (FA). Neutral lipid FA composition of animal and marine fat is strongly associated with their diet, whereas polar lipids regulate the function of membrane cells.
Iatroscan is an instrument that combines thin-layer chromatography (TLC) resolution efficacy with the capacity of quantification by flame ionization detection (FID) [
The purposes of this work were (a) to study the suitability of the Iatroscan TLC-FID analysis for rapid and complete separation and quantitation of neutral lipids (NL) into individual NL classes as well as polar lipids (PL) into individual phospholipid (PhL) classes; (b) to improve the separation capacity of GC-FID analysis of saturated and unsaturated fatty acid methyl esters (FAME) due to the nutritional and health benefits of fatty acids, especially the
The lipid standards used were cholesteryl oleate, cholesterol, octadecyl hexadecanoate, squalene, tristearoyl-glycerol, stearic acid, oleic acid, 1,3-distearoyl-glycerol, 1,2-distearoyl-glycerol, 1-monostearoyl-rac-glycerol, phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyeline standards of the Sigma Chemical Co (Sigma-Aldrich Company, Dorset, Great Britain and St. Louis, MO, USA) as well as egg yolk and mollusc muscle lipids available by our laboratory (Instrumental Food Analysis Laboratory, Department of Food Technology).
Fatty acid methyl esters standards with chain lengths from C4 to C24 from two sources were used: (a) authentic standard mixtures commercially available (Supelco 37 Component FAME Mix C4–C24, 100 mg Neat. Catalog No.: 18919-1AMP and Supelco PUFA No. 1, Marine Source, 100 mg Neat. Catalog No. 47033. Fatty acid methyl ester standards (5 mg of each) from Sigma Chemical Co: palmitic acid M-E; stearic acid M-E, oleic acid (
Lipid classes were separated on silicic acid-coated quartz rods, chromarods (Type SIII) (5 mm silica gel-coated quartz rod, Iatron Labs, Tokyo, Japan), and afterwards they were quantified using a thin layer chromatography-flame ionisation detection system. TLC-FID analysis was performed by an Iatroscan thin-layer chromatograph (Model MK-6 TLC/FID - FPD Analyser Iatron Laboratories, Tokyo, Japan) equipped with a flame ionization detector and connected to a personal computer for collecting the chromatograms. Operation conditions for the Iatroscan were 160 mL min−1 hydrogen flow, 2 L min−1 air flow, and 30 s/chromarod scan speed [
Fatty acid methyl esters (FAME) of total lipids were prepared according to the procedure described by Sinanoglou and Miniadis-Meimaroglou [
All measurements were obtained (at least) in triplicate and values were averaged and reported along with the standard deviation (S.D). All data concerning lipid and fatty acid composition were analyzed with One-Way ANOVA Post Hoc Tests and pairwise multiple comparisons were conducted with the Tukey’s honestly significant difference test. Possibilities less than 0.05 were considered statistically significant (
The present study focuses on introducing a procedure for the efficient separation and quantification of the individual neutral and polar lipid classes. Several solvent systems consisting of n-hexane, diethyl ether, petroleum ether, chloroform, methanol, acetone, formic acid, acetic acid, ammonia, and water were tested with lipid standards and egg yolk lipids in order to achieve their efficient separation. Solvent systems consisting of n-hexane-diethyl ether gave better separation of neutral from polar lipid classes than those of n-hexane-petroleum ether. Chloroform-methanol combination gave better phospholipids separation than chloroform-acetone. The presence of acetone causes the development of carotenoids associated with the phospholipids. Formic and acetic acid addition in neutral solvent systems gave better separation of triglycerides and free fatty acids than ammonia. Formic acid was preferable than acetic acid since it was more volatile and thus easily removed from the rods prior to scanning. Thus, the neutral and polar solvent systems chosen were hexane-diethyl ether with or without formic acid system combinations (HDF) and chloroform : methanol : water : with or without formic acid (CMWF) solvent system combinations, respectively.
The neutral solvent systems further examined consisted of n-hexane-diethyl ether-formic acid (n-H : DE : FA) [42 : 28 : 0.3, 55 : 20 : 1.5, 60 : 15 : 1.5, 62.6 : 6.6 : 0.8, 65 : 5 : 0.15, 66.5 : 3.5 : 1.7, and 54.9 : 3.7 : 1.4] (by vol.) as well as of n-hexane-diethyl ether (n-H : DE) [72 : 0.8 by vol.]. The presence of formic acid was found necessary in order to avoid broadening peaks. Wax ester appeared as a small peak just before sterol ester but it was incompletely separated, when present in large amount, in all the solvent system tested. The most effective separation of TG from FFA as well as of diacylglycerol isomers 1,2 and 1,3 from free sterols was achieved when a solvent system of n-H : DE : FA was used, with the ratio of n-H : DE exceeding 4 : 1. Thus, n-H : DE : FA (60 : 15 : 1.5 by vol.) (solvent A) was selected as the most appropriate solvent system for individual neutral lipid separation. A typical separation of neutral lipid standards using the Iatroscan and the above-mentioned solvent system designed to separate neutral lipids is shown in Figure
Retention time, calibration data, and reproducibility for neutral and polar lipid standards.
Solvent system | Lipid standards | Retention time | Regression equation | Correlation coefficients ( |
CV% of intraday variability | CV% of interday variability |
---|---|---|---|---|---|---|
Solvent A |
HC | 0.02–0.03 |
|
0.9994 | 1.28 | 1.69 |
Sterol esters |
0.04–0.06 |
|
1 | 1.34 | 1.76 | |
TG | 0.07–0.10 |
|
0.9984 | 2.33 | 1.83 | |
FFA | 0.11–0.12 |
|
0.9926 | 1.57 | 2.21 | |
1,3-DG | 0.16–0.18 |
|
0.9987 | 1.62 | 2.49 | |
Sterol | 0.20–0.24 |
|
1 | 2.28 | 3.46 | |
1,2-DG | 0.31–0.34 |
|
1 | 1.29 | 1.87 | |
MG | 0.36–0.39 |
|
0.9993 | 1.56 | 2.17 | |
PL | 0.42–0.45 | — | — | — | — | |
| ||||||
Solvent B |
NL | 0.02–0.08 | — | — | — | — |
PE | 0.09–0.11 |
|
0.9987 | 1.74 | 2.86 | |
PI | 0.11–0.14 |
|
0.9997 | 2.64 | 3.35 | |
PS | 0.15–0.18 |
|
0.9997 | 2.47 | 3.68 | |
l-PE | 0.19–0.20 |
|
0.9995 | 1.92 | 3.04 | |
PnL | 0.21–0.23 | — | — | — | — | |
PC | 0.24–0.30 |
|
0.9997 | 1.40 | 2.48 | |
Sphm | 0.33–0.35 |
|
0.9994 | 1.76 | 2.55 | |
l-PC | 0.37–0.40 |
|
0.9998 | 1.63 | 2.26 |
Development distance/times of development: 10 cm (1 time).
Chromatograms of (a) standard neutral lipids developed with n-hexane-diethyl ether-formic acid (60 : 15 : 1.5 by vol.), (b) standard phospholipids developed with chloroform : methanol : water (50 : 20 : 2 by vol.).
Different ratios in chloroform : methanol : water solvent system, conventionally used in TLC for the separation of polar lipids, were tested. The examined polar solvent systems consisted of chloroform : methanol : water (C : M : W) [45.2 : 22.6 : 2.2; 50 : 25 : 2.5; 45 : 20 : 2 (2 times); 45 : 20 : 2 (1 time); 48 : 22 : 1; 50 : 20 : 2.5; 50 : 20 : 2; 50 : 20 : 2.5 (5 cm) and 60 : 10 : 1 (10 cm)] (by vol.) as well as of chloroform : methanol : water : formic acid (C : M : W : FA) [45 : 25 : 2.5 : 1 and 45 : 20 : 2 : 1] (by vol.). The best separations for phospholipids were obtained with C : M : W (50 : 20 : 2, 45 : 20 : 2 and 48 : 22 : 1, by vol.), while the best separation between PI and PS was obtained with C : M : W (50 : 20 : 2, by vol.). L-PE appeared as a small peak just after PS but it was incompletely separated, when present in large amounts. As natural lipid samples contain trace amount of l-PE this peak could be easily separated from PS peak. In the same solvent system ceramide aminoethylphosphonic acid (phosphonolipid) appeared as a small peak between PS and PC when mollusc muscle lipid sample was applied in chromarods, in accordance to the findings of Sinanoglou and Miniadis-Meimaroglou [
Various multiple development solvent systems were examined, consisting of C : M : W (first development) and n-H : DE : FA (second development) [50 : 20 : 2.5 (5 cm) and 65 : 5 : 0.15 (10 cm) by vol], [50 : 20 : 2.5 (6 cm) and 65 : 5 : 0.15 (10 cm) by vol], [50 : 20 : 2.5 (7 cm) and 65 : 5 : 0.15 (10 cm) by vol], [50 : 20 : 2 (5 cm) and 65 : 5 : 0.15 (10 cm) by vol], [50 : 20 : 2 (5 cm) and 60 : 15 : 0.15 (10 cm) by vol], and [45 : 20 : 2 (5 cm) and 60 : 15 : 0.15 (10 cm) by vol]. The overlap of MG and PE peaks did not allow the quantification of these two classes of compounds when their amount was higher than 0.2
Based on the results obtained, the quantification of individual neutral and polar lipid classes was achieved using calibration curves obtained for each authentic standard by plotting peak area against lipid concentration (different concentrations: 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, and 20.0
The reproducibility of the measurement was performed from the analysis of two pure standard mixtures of neutral and polar lipids in appropriate ratios simulating the lipid composition of most food samples. Therefore, a composite standard for neutral lipids using squalene, cholesteryl oleate, tristearoyl-glycerol, cholesterol, oleic acid, 1,3-distearoyl-glycerol 1,2-distearoyl-glycerol, and 1-monostearoyl-rac-glycerol, in the ratios 1/1/5/3/1/1/1/1 by wt, as well as for the polar lipids using phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyeline in the ratios 4/2/1/1/1/l/1 by wt, was prepared. Reproducibility was tested by spotting the same standard mixture on 10 chromarods and measuring the standard deviation (SD) and the coefficient of variation (CV%). The neutral solvent system consisted of n-H : DE : FA (60 : 15 : 1.5 by vol.) while the polar solvent system consisted of C : M : W (50 : 20 : 2. by vol.). Four series of ten rods were analysed over a three-day period per mixture selecting a total lipid concentration ≤20.0
The above separation of NL and PL by Iatroscan TLC-FID offers several advantages over TLC. Higher sensitivity and better recovery were obtained, particularly for lipids present in very small amounts. TLC-FID has the additional advantage to run mixture of lipid standards in separate chromarod to check separation in each analysis. The neutral and polar lipid separation in individual NL and PhL classes is completed in a reasonable time of approximately 1 and 0.5 h, respectively, per 10 analyses. Complex lipid samples do not require pretreatment and even a small sample size (few
The procedure for fatty acid methyl esters (FAME) analysis using the GC conditions as modified in this study and described in the Material and Methods paragraph, was applied to commercially FAME standards. Figure
FAME retention times and response factors.
A/A | FAME | Rt (min) | Rf (split ratio1 : 2) |
---|---|---|---|
1 | C4:0 | 4.200–4.405 |
|
2 | C6:0 | 4.580–4.760 |
|
3 | C8:0 | 5.438–5.808 |
|
4 | C10:0 | 6.897–7.158 |
|
5 | C10:1 | 7.325–7.579 |
|
6 | C11:0 | 7.877–8.088 |
|
7 | C12:0 | 9.064–9.251 |
|
8 | C13:0 | 10.484–10.641 |
|
9 | C14:0 | 12.168–12.398 |
|
10 | C14:1 | 12.954–13.131 |
|
11 | Iso-C15:0 | 13.556–13.685 |
|
12 | Anteiso-C15:0 | 13.782–13.956 |
|
13 | C15:0 | 14.148–14.354 |
|
14 | C15:1 |
15.064–15.274 |
|
15 | C16:0 | 16.400–16.791 |
|
16 | Iso-C16:0 | 16.978–17.162 |
|
17 | C16:1 |
17.212–17.394 |
|
18 | Iso-C17:0 | 18.465–18.588 |
|
19 | Anteiso-C17:0 | 18.612–18.688 |
|
20 | Cyclo-C17:0 | 18.705–18.855 |
|
21 | C17:0 | 18.948–19.100 |
|
22 | C17:1 |
19.728–19.994 |
|
23 | C18:0 | 21,573–22,000 |
|
24 | C18:1 |
22.000–22.285 |
|
25 | C18:1 |
22.285–22.668 |
|
26 | C18:1 |
22.749–22.909 |
|
27 | C18:2 |
23.011–23.301 |
|
28 | C18:2 |
23.314–23.301 |
|
29 | C18:2 |
23.671–23.951 |
|
30 | C18:3 |
24.488–24.764 |
|
31 | C18:3 |
25.434–25.721 |
|
32 | C18:4 |
26.305–26.515 |
|
33 | C19:0 | 27.336–27.492 |
|
34 | C20:0 | 27.492–27.653 |
|
35 | C20:1 |
28.042–28.485 |
|
36 | C20:2 |
29.660–29.867 |
|
37 | C20:3 |
30.528–30.660 |
|
38 | C20:4 |
31.235–31.508 |
|
39 | C20:3 |
31.888–32.037 |
|
40 | C21:0 | 32.964–33.282 |
|
41 | C20:5 |
33.675–34.151 |
|
42 | C22:0 | 34.329–34.598 |
|
43 | C22:1 |
36.285–36.361 |
|
44 | C22:1 |
36.534–36.892 |
|
45 | C22:2 |
37.633–37.753 |
|
46 | C23:0 | 37.979–38.308 |
|
47 | C22:4 |
38.527–38.743 |
|
48 | C22:5 |
39.855–40.198 |
|
49 | C22:5 |
40.386–40.822 |
|
50 | C24:0 | 42.544–42.703 |
|
51 | C22:6 |
43.255–44.162 |
|
52 | C24:1 |
44.162–44.392 |
|
Typical chromatogram showing the separation of FAME standards Supelco 37 Component FAME Mix C4–C24, (Merck, Darmstadt, Germany) on a DB-23 capillary column (60 m × 0.25 mm i.d. 0.15
In order to validate the method in terms of linearity, standard solutions containing different concentrations of 37 Component FAME Mix were prepared and subjected to GC analysis. Injection of solutions containing ten different concentrations of 37 Component FAME Mix in n-hexane (ranging from 0.5 to 5.0, from 5.0 to 20.0, and from 10.0 to 30.0
The amounts of FAME in the studied samples were then calculated via the individual FAME peak area and the Rf.
FAME response factors (Rfs) for split ratio 1 : 2 are presented in Table
Dodds et al. [
The detection limit (DL) and quantitation limit (QL) were obtained for a signal-to-noise (S/N) ratio of 3 and 10, respectively. Four concentrations of 0.001, 0.002, 0.005, and 0.01
Three different concentrations of 37 Component FAME Mix (10.0, 15.0, and 20.0
The above separation of FAME by GC-FID offers several advantages, such as high repeatability and reproducibility of retention times and high precision in quantitation based on peak area measurements. Several research studies confirmed the stationary phase polarity and column length impact to the resolution of fatty acids isomers [
The combination of Iatroscan TLC-FID and GC-FID methodologies has been applied to several food samples to identify neutral and polar lipid profiles, composition and fatty acid content of total fat extracted. A summary of these applications is presented in Table
Summary of applications of the combined Iatroscan TLC-FID and GC-FID methodologies.
Lipid sample origin | Iatroscan TLC-FID | GC-FID | Reference | |||
---|---|---|---|---|---|---|
Neutral lipids | Polar lipids | Fatty acids (FA) | ||||
Ostrich ( |
30 FA | |||||
Turkey ( |
33 FA | |||||
Egg yolk | Quail ( |
TG, FFA, Chol, 1,3-DG, MG | PE, PI, PC, Sphm, l-PC | 33 FA | Sinanoglou et al. [ | |
Duck ( |
30 FA | |||||
Animal origin | Goose ( |
30 FA | ||||
Medium-growth broilers | Intramuscular fat |
TG, FFA, Chol, 1,3-DG, MG | PE, PS, PC, Sphm, l-PC | 35 FA |
Sinanoglou et al. [ | |
Lamb/kid | Intramuscular fat |
TG, FFA, Chol, DG, MG |
PE, PS, PC, Sphm, l-PC |
33–36 FA |
Sinanoglou et al. [ | |
Lamb milk/creme | TG, Chol, MG | 35–40 FA | Unpublished data | |||
| ||||||
Langoustine |
Muscle—cephalothorax | Waxes, sterol esters, TG, FFA, Chol, 1,3-DG, MG | 13–15 FA |
Tsape et al. [ | ||
Shrimp |
Muscle—cephalothorax | Waxes, sterol esters, TG, FFA, Chol, 1,3-DG, MG | PE, PI, PS, PnL, PC, Sphm, l-PC | 23–33 FA | Tsape et al. [ | |
Marine origin | Red porgy wild |
|||||
Greater weever ( |
Muscle | Cardiolipine, PE, PI, PS, PC, Sphm, l-PC | 29 FA | Loukas et al. [ | ||
Piper gurnard ( |
||||||
Picarel |
TG, FFA, Chol, 1,3-DG, MG | PE, PI, PS, PC, Sphm, l-PC | 38 FA | Zervou et al. [ | ||
| ||||||
Plant origin | Sesame seeds ( |
TG, FFA, Chol, 1,3-DG, MG | PA, PE, PI, PS, PC, Sphm, l-PC | 18 FA | Zoumpoulakis et al. [ | |
| ||||||
Mushrooms |
|
Fruit body | 28 FA | |||
Mycelium | 22 FA |
Papaspyridi et al. [ | ||||
|
Fruit body | 29 FA | ||||
Mycelium | 19 FA |
Besides, for GC-FID analysis, the efficiency of the selected column and temperature programme for separation of the C18 fatty acids and their
The authors declare no conflict of interests.