1.14: Separation of the Phosphatidylcholines Using Reverse Phase HPLC - Biology

1.14: Separation of the Phosphatidylcholines Using Reverse Phase HPLC - Biology

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14.1 Learning Objective

This laboratory has 2 goals, (1) to learn more of membrane lipid structures by working with phosphatidylcholines and (2) to learn the basics of an especially important high performance liquid chromatography (HPLC) technique, reverse phase HPLC. You should use your knowledge of phosphatidylcholine structures to rationalize the elution pattern from the HPLC.

14.2 Phosphatidylcholines

Phosphatidylcholine is an important class of lipids (hydrophobic biochemicals). This class is one of the primary constituents of the biological membrane. Phosphatidylcholines have a common structure. To build a phosphatidylcholine, start with glycerol. Other components are connected to glycerol using ester bonds. Two fatty acids (long chain carboxylic acids) are esterified to the top two positions of glycerol. The third position contains a phosphate and a choline. Each phosphatidylcholine differs from others of its class based on the molecular characteristics of the fatty acid chains (Fig. 14.1).

14.3 High Performance Liquid Chromatography (HPLC)

Separation of phosphatidylcholines is difficult, but can be done using high performance liquid chromatography (HPLC). This separation technique depends on passing a solution (the mobile phase) through a column packed with very tiny particles (the stationary phase). Some solutes are attracted strongly to these particles, and travel through the column slowly. These solutes stick to a particle for a certain length of time and then “hop” to the next particle. Compared with the motion of the mobile phase, these solutes are retarded. Other solutes are only attracted to the particles weakly and so can travel through the column quickly.

The differential movement of solutes leads to the separation of the solute. This is often shown as a chromatogram (Fig. 14.2).

You will be using reverse phase HPLC. The particles in this technique are made of silica (sand) that has been coated with alkane chains. Solutes that are more hydrophobic are more strongly attracted to the stationary phase and move more slowly through the column. You will be separating five phosphatidylcholine compounds with very similar structures (Fig. 14.3).

Phosphatidylcholines with the longer fatty acid chains are more strongly attracted to the stationary phase. The length of the fatty acid chains depends on:

– the number of carbons,

– the presence of cis double bonds. Each cis double bond makes the chain act as if it were one to two carbons shorter (less hydrophobic).

Which of the five phosphatidylcholines would you predict to be attracted most to the stationary phase? Which would you predict will be attracted least? Based on the PC structure, predict the order of elution from the column.

14.4 Quantifying Chromatography

The success of a separation can be measured in several different ways. First, quicker separation tends to be better, because the experimenter doesn’t have to wait for her/ his results too long. For each solute a retention time is measured. This is the elapsed time from the beginning to when the solute peak leaves the column. Typically, this time is reported relative to the quickest elution time (the time it takes for solvent to pass through the column, the void time). So, the quickness of separation is measured by relative retention or capacity factor (k´).

Using the chromatogram in Fig. 14.2, Peak A has a retention time of 9.3 min. The void time for this chromatogram is 2.0 min.

Peak A is retained on the column 3.6-times longer than the mobile phase.

Another measure of success is to monitor the shape of each peak. Sharp peaks mean a better separation. This is measured by determining the width of the peak relative to how long the peak is retained on the column.

The Peak A from Fig. 14.2 has a retention time of 9.3 min with a peak width of 0.8 min.

[mathrm{N}=16left(frac{9.3 mathrm{~min}}{0.8 mathrm{~min}} ight)^{2}=2162]

A well-done separation will give efficiencies in the thousands.

A third measure of success is to quantify how much separation occurs between neighboring peaks. This selectivity is calculated as the ratio of capacity factors

Peak A has a k´= 3.6 while Peak B has a k´= 4.3. This means, this separation between Peak A and Peak B has a selectivity of 1.2. Peak B is retained 20% longer than Peak A. A good separation will give selectivity values of greater than 1.1.


Reagents and equipment needs are calculated per six student teams. There is ~20% excess included.

Equipment/glassware needed:

  1. Standard HPLC system 1 per 2 student teams
  2. C-18 reverse phase HPLC column

Reagents needed:

  1. 98% methanol
  2. 100 μl of phosphatidylcholines mix dissolved in methanol. Stock concentrations for each phosphatidylcholine in the mix are listed below
    1. DMPC 10 mg/ml
    2. DPoPC 5 mg/ml
    3. DLPC 1 mg/ml
    4. POPC 5 mg/ml
    5. DOPC 5 mg/ml

Experimental procedure:

  1. A standard analytical, reverse phase HPLC column (C-18) is equilibrated with 98% methanol - 2% water. A flow rate of 1 ml/min is convenient.
  2. Each separation uses 10 μl of a mixed phosphatidylcholine sample.
  3. The sample contains 10 mg/ml DMPC, 5 mg/ml DPoPC, 1 mg/ml DLPC, 5 mg/ml POPC, 5 mg/ml DOPC in methanol.
  4. Each separation requires about forty minutes.
  5. Follow your instructor’s directions concerning operation of the HPLC chromatograph.

Data Analysis:

  1. Calculate the capacity factor (relative retention) for each phosphatidylcholine.
  2. Determine the column efficiency (N) calculated using the DLPC peak.
  3. Calculate the selectivity (α) between (a) DMPC vs. DPoPC, (b) DPoPC vs. DLPC, (c) DLPC vs. POPC and (d) POPC vs. DOPC.

Notes to Instructor

This laboratory is scheduled to maximize use of a limited number of chromatographs. At the authors’ institution, we use three HPLC machines simultaneously. Two student teams (each team composed of a student pair) are assigned to each chromatograph. While one team runs the chromatography, the other team is completing an in-lab HPLC problem set. Thus, by the end of the period, all teams have completed a chromatographic trial and practiced the common calculations needed to analyze a chromatogram.

HPLC of Lipids Prelab

1. Draw the structure of each phosphatidyl choline that you are going to separate during lab (there are five)!

2. Circle the hydrophobic part of each molecule!

3. Rank these molecules based on hydrophobicity from least (5) to most hydrophobic (1)!

4. Which of the five PCs would you predict to be attracted most to the stationary phase? Which would you predict will be attracted least?

5. Based on the PC structure, predict the order of elution from the column.

HPLC of PhosphatidylcholinesLab Report Outline and Point Distribution


1. Several sentences defining the goal/purpose of this experiment. (3 pts.)


  1. A copy of your chromatogram with each peak labeled with a specific phosphatidylcholine. (10 pts.)

Results (please show all calculations)

  1. The capacity factor (relative retention) for each phosphatidylcholine. (10 pts.)
  2. The column efficiency (N) calculated using the DLPC peak. (4 pts.)
  3. The selectivity (α) between (a) DMPC vs. POPC, and (d) POPC vs. DOPC. (8 pts.)


  1. Which phosphatidylcholines are cleanly separable on this column. Briefly explain. (5 pts.)
  2. Problems (10 pts.)

HPLC Problem Set

(Courtesy of Dikma Technologies. Chromatorex is a registered trademark of Fuji Silysia Chemical Ltd. Dikma Technologies Inc. is not affiliated with the above company.

1. (5 pts.) The chromatogram from Chromatorex-SMB appears not as good as the chromatogram from Inspire. Calculate the column efficiencies (N) based on peak 3. (Use a ruler and the conversion, 1 minute/6 mm.) Does this agree with the conclusion in the first sentence? Briefly explain.

(Courtesy of SIELC Technologies)

2. (5 pts.)By just looking at the chromatograms, rank them from best separation to worst separation. Using the two chromatograms that are measurable, determine retention times for each peak. (Estimate times to the nearest 0.1 minute.) Calculate capacity factors for each peak using 1.1 min as the void time. Then, calculate selectivity factors (α) for Peak 1 vs. Peak 2 and for Peak 2 vs. Peak 3. Do the selectivity factors agree with your ranking? Briefly explain.

1.14: Separation of the Phosphatidylcholines Using Reverse Phase HPLC - Biology

High performance liquid chromatography methods were established for separation of alkenylacyl, alkylacyl, and diacyl acetylglycerols derived from ethanolamine glycerophospholipids (EGP) and for separation of the individual molecular species from each of the separated classes. The EGP were isolated from bovine brain, hydrolyzed with phospholipase C, and acetylated with acetic anhydride. The three classes of diradylacetylglycerols were separated quantitatively on a mu Porasil silica column. Individual classes were further fractionated on a Zorbax ODS reverse phase column. By gas--liquid chromatographic quantitation of each peak, 29-33 different molecular species were identified within each class. For alkenylacyl-GPE, the major species were 18:1-18:1, 21.8%, and 16:0-18:1, 14.8%. Polyenoic fatty acids predominated at the 2-position of diacyl-GPE. The major species were 18:0-22:6 (n-3), 25.5%, and 18:0-20:4 (n-6), 15.8%. Three species of alkylacyl-GPE, 18:0-20:6 (n-3), 16:0-22:4 (n-6), and 18:0-22:4 (n-6), each accounted for 10%.


The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.

RP-hPLC/ESI MS determination of acyl chain positions in phospholipids

Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences (UIPS) and Bijvoet Center for Biomolecular Research, Utrecht University, PO Box 80 082, 3508 TB Utrecht, The Netherlands

OctoPlus B.V., Zernikedreef 12, 2333 CL Leiden, The Netherlands

Laboratory of Veterinary Biochemistry and Institute of Biomembranes, Utrecht University, PO Box 80 176, 3508 TD Utrecht, The Netherlands

Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences (UIPS) and Bijvoet Center for Biomolecular Research, Utrecht University, PO Box 80 082, 3508 TB Utrecht, The Netherlands

OctoPlus B.V., Zernikedreef 12, 2333 CL Leiden, The Netherlands


Since phospholipids are used as excipients in many pharmaceutical products, there is a need for validated strategies to characterize the phospholipid constituents. We describe a reversed phase HPLC/electrospray ionization mass spectrometry method that is an alternative to the laborious phospholipase A2 treatment commonly used to determine the acyl chain positions in phospholipids. This reversed phase HPLC/electrospray mass spectrometry system (a) shows good chromatographic resolution for phosphatidylcholine molecular species, (b) allows determination of the composition of complex mixtures of phosphatidylcholines, and, most important, (c) allows unequivocal assignment of the positions of the acyl chains on the glycerol backbone in mixtures of POPC and OPPC and in mixtures of POPE and OPPE.

Combined Reversed Phase HPLC, Mass Spectrometry, and NMR Spectroscopy for a Fast Separation and Efficient Identification of Phosphatidylcholines

In respect of the manifold involvement of lipids in biochemical processes, the analysis of intact and underivatised lipids of body fluids as well as cell and tissue extracts is still a challenging task, if detailed molecular information is required. Therefore, the advantage of combined use of high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy will be shown analyzing three different types of extracts of the ubiquitous membrane component phosphatidylcholine. At first, different reversed phase modifications were tested on phosphatidylcholines (PC) with the same effective carbon number (ECN) for their applicability in lipid analysis. The results were taken to improve the separation of three natural PC extract types and a new reversed phase (RP)-HPLC method was developed. The individual species were characterized by one- and two-dimensional NMR and positive or negative ion mode quadrupole time of flight (q-TOF)-MS as well as MS/MS techniques. Furthermore, ion suppression effects during electrospray ionisation (ESI), difficulties, limits, and advantages of the individual analytical techniques are addressed.

1. Introduction

The analysis of native and underivatized lipids within body fluids as well as cell and tissue extracts is still a challenging task, in particular, if the molecular structure of individual components needs to be identified in decently short time. The lipid composition consists of different main classes such as fatty acids, neutral lipids, and lipids with positively or negatively charged head groups with manifold subclasses of structural diversity. Variations within the lipid composition were attributed to different pathologies such as neoplastic and neurodegenerative diseases, diabetes mellitus, and many others. Furthermore, some lipid classes are involved in cell death (apoptosis, necrosis), cellular signaling and are precursors for lysophospholipids (i.e., lysophosphatidylcholine), diacylglycerols, and phosphatic and arachidonic acid [1–25].

1,2-Diacyl-sn-glycero-3-phosphatidylcholine (PC) represents a major constituent of cell membranes. It consists of the polar head group phosphorylcholine attached to the sn-3 position of glycerol and differing saturated and unsaturated fatty acids esterified to the sn-1 and sn-2 position, whereby fatty acids in position sn-1 are preferentially saturated as a rule. Numerous studies dealt with PCs in the past because of their utmost biochemical and clinical importance and many different analytical techniques have been proposed to get an insight into metabolic turnover or to characterize pathophysiological deviations of the native lipid composition. Most of these techniques suffer from various drawbacks as being time-consuming, insensitive, destructive, or not related to individual substructures. Gas chromatography (GC) [26–28], thin layer chromatography (TLC) [29–31], and high-performance liquid chromatography (HPLC) [32–37] are commonly used for lipid analysis. GC-based techniques are quantitative but require time-consuming sample preparation techniques. GC is often used in combination with TLC for the lipid class separation. The spots on a TLC plate are scratched out and their fatty acid residues are analyzed upon derivatization into a volatile substrate and recorded by GC. However, the precise molecular structure of an individual lipid is lost because of the preceding hydrolysis of the lipids. Enzymatic cleavage of the ester bond using phospholipases allows a successive hydrolysis of the sn-2 and sn-1 fatty acid, but it is rather time-consuming due to intense laboratory work and already minor contamination of the enzyme leads to false results. HPLC offers the separation of lipid classes using the normal phase mode (NP) and additionally the separation according to the different fatty acid residues of an individual lipid in the reversed phase mode (RP). In this case, a successful separation depends distinctly on the appropriate selection of the stationary phase. Alternatively, MS-based techniques are widely used, as they are fast, sensitive and require only minor sample preparation [38]. The use of high resolution MS systems give access to the molecular formula. In addition, characteristic fragmentations identify the lipid class and molecular structure. When coupled with a HPLC-system their selectivity is much higher and benefits from both techniques. NMR spectroscopy is capable to measure intact biomaterials nondestructively without any preceding derivatization. Especially 31 P-NMR is well-suited to quantify phospholipid class analysis and needs only less sample preparation [39–44]. Again only minor information is obtained with respect to the fatty acid residues. 1 H-NMR measurements are also widely used, as they contain more information about the fatty acids in general, but the connection to the glycerol backbone is missing due to massive signal overlap. 2D-NMR involving the 13 C nucleus provides a lot more resolution and more information about individual species, but the low NMR sensitivity of the 13 C isotope prevents a fast and wide application of this technique in a routine analysis [44–48].

This paper presents an efficient RP-HPLC setup to separate phosphatidylcholines, which ultimately will be extendable to separate other polar phospholipids. Subsequently the HPLC tool is combined with the highly informative molecular assignment potentials of MS and NMR [49]. Five different types of silica-based reversed phase modifications were tested with respect to their capability to separate lipids containing fatty acids with an equivalent carbon number (ECN), which is the number of carbon atoms within a fatty acid chain minus twice the number of double bonds. The extension of a lipid by a C=C double bond will not change the hydrophobicity. The performance of all columns was tested on a mixture of five PCs with the same ECN whereas two of them are even constitutional isomers concerning the 1,2-positions of glycerol, which hampers the separation even more.

Then, the HPLC column with best performance was used to achieve an efficient baseline separation of three native PC extracts (soy bean, bovine brain, and egg yolk). Furthermore, the MS fragmentation behavior in the positive and negative ion mode is investigated for individual PCs to identify characteristic fragmentation patterns for this lipid class and its fatty acid residues. 1D and 2D high-resolution NMR spectra were also acquired to confirm the molecular structure.

2. Material and Methods

2.1. Chemicals

Methanol-d4 and deuterated chloroform, methanol (LC-MS grade), all fatty acids, the test mixture compounds dipalmitoyl-phosphatidylcholine (DPPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), oleoyl-palmitoyl-phosphatidylcholine (OPPC), dioleoyl-phosphatidylcholine (DOPC), stearoyl-linoleoyl-phosphatidylcholine (SLPC), and also the soy bean, bovine brain, and egg yolk extracts were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The double distilled water was taken from the in-house system.

2.2. High-Performance Liquid Chromatography

A HP 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) was used. The injection volume was 3

L of the standard prepared in methanol. Five columns with different stationary phases were tested with respect to their separation performance for lipid analysis: (1) type A silica-based endcapped C18 (Nucleosil 100-5 C18, 250

3 mm), (2) type A silica-based phenyl (Nucleosil 100-5 C6H5, 250 4 mm), (3) type B silica-based high density C18 (Nucleodur C18 Gravity, 5 m, 250 3 mm), (4) type B silica-based polymer/cross linked C18 (Nucleodur C18 Isis, 5 m, 250 3 mm), (5) type B silica-based mixed mode phenyl/C18 (Nucleodur Sphinx RP, 5 m, 250 3 mm).

All HPLC columns and materials were a kind gift of Macherey-Nagel (Düren, Germany).

The 3 mm columns were operated at flow rate of 0.6 mL/min and the 4 mm column at 1 mL/min. The mobile phase was optimized by adapting the methanol content in different runs between 90% and 100% for the alkyl phases and between 80% and 100% for the phenyl phase with respect to the hydrophobic interaction of the analytes with the RP packing.

An 8 mm Nucleodur Sphinx RP was operated under isocratic conditions at 4.1 mL/min flow with a mobile phase consisting of methanol and water (90 : 10) for the semi preparative approach. To collect the individual species for NMR measurements a Gilson 215 liquid handler (Gilson International B.V., Bad Camberg, Germany) was used. The column temperature was kept at 4

2.3. Mass Spectrometry

An esquire LC iontrap system (Bruker Daltonik GmbH, Bremen, Germany) was used for mass spectrometric detection for positive and negative ion mode mass and MS/MS spectra of each PC compound were recorded. The capillary voltage was set to

3800 V and the end plate offset to 500 V in positive ion mode. For the HPLC the nebulizer gas was set to 40 psi, dry gas and dry heat were set to 10 L/min and 30 C, respectively. In case of direct infusion via a syringe pump, the dry and nebulizer gases were reduced to 5 L/min and 5 psi, respectively. The collision energy for MS/MS experiments was optimized with respect to the precursor ion stability.

A micrOTOF-Q-equipped with the Apollo ESI ion source (Bruker Daltonik GmbH, Bremen, Germany) was used for precision mass detection. The capillary voltage was set to 4500 V and the end plate offset to 500 V in negative ion mode. The nebulizer gas was set to 0.4 bar, dry gas and dry heat were set to 4 L/min and 20 C, respectively. For MS/MS experiments the collision energy of the quadrupole was 42 eV/z. The molecular formula was generated by matching high mass accuracy and isotopic pattern (SigmaFit, Bruker Daltonik GmbH, Bremen, Germany).

2.4. Nuclear Magnetic Resonance

All samples were stored at 8 C before the measurements. In case of dissolved samples, the solvents were evaporated by a gentle stream of nitrogen and redissolved in CDCl3/ CD3OD (2 : 1). 1D ( 1 H, 13 C) and high-resolution 2D (HSQC, HSQC-TOCSY, HMBC) NMR spectra with a digital resolution of 1k data points in F1 and 4k data point in F2 dimension of each PC species were acquired on a Bruker DRX 600 MHz NMR spectrometer equipped with 5 mm TXI probe (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Germany).

3. Results

3.1. High-Performance Liquid Chromatography

A comparison of five different reversed phase columns revealed the following behavior: The separation of the test mixture on type A silica-based materials showed only poor results for all PC compounds. Although, it seems that the Nucleosil material separates all peaks very well (see Table 1), the extreme peak broadening and a distinct tailing spoils the pretended peak separation. In contrast, the type B silica based materials separated DPPC, DOPC, SLPC, and POPC or OPPC very well. However, the two lipid isomers POPC and OPPC were only well separated (Table 1) on the polymer cross link RP packing (ISIS). With all RP materials it was possible to separate lipids containing two monounsaturated fatty acids from lipids with one or two saturated or one polyunsaturated fatty acid. The shortest separation times with sharp chromatographic peaks were achieved by the mixed mode stationary phase (Sphinx). Therefore, this stationary phase was selected to separate the individual compounds within the lipid extracts of natural sources.


Sphingomyelins were characterized using a combination of a novel isocratic reversed-phase HPLC method with electrospray time-of-flight mass spectrometric detection and optional online MS/MS. The constitution of the sphingomyelins is determined by MS/MS experiments. Baseline separation of 17 compounds of a bovine brain extract (2 main compounds and 15 minor or trace compounds) was achieved with a mobile phase consisting of methanol, 2-propanol, THF, and water on a RP-18-phenyl column. In parallel, the HPLC fraction were sampled to a 600-MHz NMR spectrometer to acquire 1D and 2D NMR spectra and to elucidate the molecular structure of individual sphingomyelin components.

To whom correspondence should be addressed. Telephone: 0049-421-218-2818. Fax: 0049-421-218-4264. E-mail: [email protected]


The use of liquid chromatography (LC) to quantify the phlorotannin compositions in macroalgal extracts is limited by the lack of commercially available standards. The only standard for calibration that is commercially available is the monomer phloroglucinol.

Koivikko et al. measured the concentration of phlorotannins through integration of peaks in a crude extract of Fucus vesiculosus. 38 The F-C assay was used to calculate the TPC of these compounds and then a Pearson correlation coefficient was calculated between the individual traces of the chromatogram and the contents of total phlorotannins. Further statistical analysis was performed to assess how well the variation in the phlorotannin chromatography profile can explain the variation of the content of total phlorotannins by conducting a multiple regression analysis. This attempt at quantification is however not complete as the area of the peak in the chromatographic profile is affected by the sensitivity of the compound to detection and the stability of the compounds under analytical conditions. 38 Therefore further research needs to be performed with characterised standards in order to fully develop qualitative methods of high-performance liquid chromatography (HPLC) analysis.

1.14: Separation of the Phosphatidylcholines Using Reverse Phase HPLC - Biology

a Institute of Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria
E-mail: [email protected]
Tel: +43 316 380 5318


Amanita muscaria, also known as the fly agaric mushroom, can accumulate vanadium (V), with up to several hundred mg V kg −1 dry mass. It is long known that V is present in A. muscaria as a complex called amavadin, but methods for the investigation of the distribution and biosynthesis of amavadin in mushrooms are missing. Here, we describe the development of the first sensitive method for the determination of amavadin and other V-containing compounds in environmental samples by employing high performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICPMS). A strong anion-exchange column serves as the stationary phase, and the mobile phase consists of an aqueous ammonium citrate buffer and ethylenediaminetetraacetate (EDTA). The concentration and pH of the mobile phase as well as the column temperature were evaluated to optimize the separation. With the final method, amavadin is eluted in less than 17 minutes, and its limit of detection is 0.05 μg V L −1 . Moreover, the compound's two isomers are separated from each other and can be quantified independently. The method was applied to extracts of fruit-body samples of A. muscaria. The extraction efficiency was 74 ± 12%, and amavadin accounted for 75–96% of the extracted V. In addition, significant concentrations of other V species could be detected, which have never been described before. Our results demonstrate that V speciation in mushrooms is more complex than assumed until now and that more in-depth investigations on this matter are needed. The developed method enables the investigation of organic and inorganic V species in the environment, even at low concentrations.


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2.1 Materials

The standards 3-hydroxybutyric acid (3-OH-FA (4:0)) sodium salt, (±)−3-hydroxyhexanoic acid (3-OH-FA (6:0)), (±)−3-hydroxyoctanoic acid (3-OH-FA (8:0)), (±)−3-hydroxydecanoic acid (3-OH-FA (10:0)), (±)−3-hydroxydodecanoic acid (3-OH-FA (12:0)), (±)−3-hydroxy myristic acid (3-OH-FA (14:0)), and rhamnolipid (R-95), di-rhamnolipid dominant (Rha), were obtained from Sigma Aldrich (Steinheim, Germany). Solvents and additives used for MS-detection were of LC-MS grade. Methanol (MeOH), acetonitrile (ACN), and acetic acid (AcOH) were obtained from Carl Roth (Karlsruhe, Germany).

2.2 Sample preparation of standards

Due to differing solubility in water, as a consequence of varying chain length, 3-OH-FA (4:0), 3-OH-FA (6:0), and 3-OH-FA (8:0) were dissolved in H2O, while 3-OH-FA (10:0), 3-OH-FA (12:0), and 3-OH-FA (14:0) were dissolved in MeOH/H2O (6:4, v/v), both at a concentration of 2 μg/mL.

2.3 Rhamnolipid hydrolysis

For acidic hydrolysis, 5 mg of rhamnolipid (R-95) was suspended in 0.5 mL 2.7 M H2SO4 in a screw-capped glass vial. A volume of 0.5 mL of CHCl3 was added and the obtained biphasic system was heated at 110°C for 140 min. The chloroform layer containing the fatty acid was collected, evaporated to dryness, and subsequently, solubilized in 1 mL MeOH.

For alkaline hydrolysis, a stock solution of 5 mg of rhamnolipid (R-95) in 0.5 mL MeOH (10 mg/mL) was prepared. Stock solution (50 μL) and a methanolic solution of 2N NaOH (50 μL) were each added to 900 μL of a solution of THF/MeOH (9:1, v/v) and stirred for 2 h at room temperature (≈25°C). The solvents were then removed under vacuum, the residue diluted with 200 μL of water and acidified with 0.1 M HCl to pH 2–3. The solution was then extracted thrice with 200 μL ethyl acetate, the combined organic layers evaporated to dryness and reconstituted with 100 μL MeOH/H2O (3:7, v/v). The solution was diluted tenfold (H2O) for LC-MS analysis.

2.4 Lipopeptide hydrolysis

The lipopeptide was first dissolved in MeOH to obtain the stock solution (10 mg/mL). An aliquot of 50 μL (corresponding to 500 μg) was added up to 1 mL with a solution of 6 M deuterated hydrochloric acid (DCl/D2O, 1:1, v/v) in a screw-capped glass vial and heated for 24 h at 110°C. The hydrochloric acid was evaporated in an EZ-2 high performance evaporator from GeneVac (Ipswich, UK). The residue was extracted with 200 μL of a mixture of water and chloroform in a ratio of 1:1 (v/v). The chloroform layer containing the 3-hydroxyalkanoic acid was evaporated to dryness using the Genevac, and the residue reconstituted with 100 μL MeOH. The solution was dissolved tenfold with H2O for RP and MeOH for HILIC measurements. The aqueous layer was likewise evaporated to dryness and used for amino acid analysis (reported elsewhere).

2.5 Instrumentation

Chiral chromatographic separation was performed on an Agilent 1290 Infinity UHPLC system (Waldbronn, Germany) equipped with a binary pump (G4220A), a column thermostat (G1316A), and a PAL autosampler (CTC Analytics AG, Switzerland). The separations were performed on a CHIRALPAK IA-U column (100 × 3.0 mm, 1.6 μm). The mobile phases comprised water (MP-A) and acetonitrile (MP-B), both containing 0.1% (v/v) acetic acid. The following gradient was applied if not otherwise stated: 0–2 min 10% MP-B, 2–20 min 10–100% MP-B, 20–22 min 100% MP-B, 22-22.1 min 100–10% MP-B, and 22.1-25 min 10% MP-B. The flow rate was 300 μL/min, the column temperature 40°C, and the injection volume 10 μL.

MS detection was performed on an AB SCIEX API 4000 MS/MS mass spectrometer equipped with a TurboIonSpray (SCIEX, Ontario, Canada) in selected reaction monitoring (SRM) mode. The parameters of the selected reaction monitoring transitions, including dwell time, collision energy, and delustering potential (DP), were optimized for each compound individually and are displayed in Table 1. The total cycle time was 385 ms. All measurements were run in negative polarity mode. The cell exit potential was set to −15 V, the entrance potential to −10 V, the ion source voltage to −4500 V, the temperature to 400°C, the nebulizer gas and heater gas pressures to 30 psi, the curtain gas to 35 psi, and the collisionally activated dissociation gas to 6 psi. PeakView 2.2 software was used for data analysis.

Name Q1 Q3 Dwell time (ms) CE DP
3-OH-FA (4:0) 103 59.1 50 −15 −80
3-OH-FA (6:0) 131.1 59.1 50 −15 −80
3-OH-FA (8:0) 159.1 59.1 50 −15 −80
3-OH-FA (10:0) 187.1 59.1 50 −20 −80
3-OH-FA (12:0) 215.2 59.1 50 −20 −80
3-OH-FA (14:0) 243.2 59.1 50 −20 −80
3-OH-FA (12:0) [M+1-H] – 132.1 59.1 50 −15 −80
3-OH-FA (14:0) [M+1-H] – 160.1 59.1 50 −15 −80

Chromatographic Methods in the Separation of Long-Chain Mono- and Polyunsaturated Fatty Acids

This review presents various chromatographic systems, TLC, HPLC, GC, and also SFC, developed for identification and accurate quantification of long-chain mono- and polyunsaturated fatty acids from different samples with emphasis on selected literature which was published during last decade. Almost all the aspects such as preseparation step of fatty acids (cis and trans), stationary phase, solvent system, and detection mode are discussed.

1. Introduction

Long-chain fatty acids (LC-FA) are organic compounds in which the hydrocarbon chain length may vary from 10 to 30 carbons. The hydrocarbon chain can be saturated or unsaturated (contains one or more double bonds). Based on the number of double bonds, unsaturated fatty acids are classified into the following groups [1, 2]: (i) monounsaturated fatty acids (monoenoic acids, MUFA), containing one double bond, for example, oleic acid, (ii) polyunsaturated fatty acids (polyenoic acids, PUFA), having two or more double bonds, for example, γ-linolenic acid, (iii) eicosanoids, which are derived from polyenoic fatty acids, for example, prostaglandins.

Recent literature data indicate that both monounsaturated and polyunsaturated fatty acids are biological important compounds which play a significant role for the living organisms [3–17]. Human feeding studies during the last ten years demonstrate that PUFA as well as MUFA are the main components of cholesterol-lowering diet [4, 7, 9]. Moreover, there has been a much interest in the effect of MUFA and PUFA on immune and inflammatory system [13]. Among various monounsaturated fatty acids, the most popular is oleic acid (C18:1n-9). It is found in plants (e.g., olive oil), animals, and microorganisms. Olive oil consumption has benefit for colon and breast cancer prevention [8]. The current studies show that oleic acid plays important role in prevention of coronary disease (ability to reduce LDL-cholesterol) [7, 9]. Polyunsaturated fatty acids similar to monounsaturated fatty acids are widely distributed in nature [18]. There are three classes of unsaturated fatty acids common in human tissues [5]: the ω-3 (n-3 PUFA), ω-6 (n-6 PUFA), and ω-9 (n-9 PUFA) fatty acids. To the group of discussed unsaturated fatty acids belongs also demospongic acid, a mixture of very long-chain fatty acids, mainly C24–C30 with the atypical 5,9-diunsaturation system. It exists in microorganisms, marine invertebrates, and terrestrial plants [19]. The main sources of omega-3 are fishes, some plants, and green algae [20]. Green oleaginous algae are the potential source of the following ω-3 acids: eicosapentaenoic (EPA), docosahexaenoic (DHA), and also arachidonic acid (AA) from ω-6 group [21–23]. Omega-6 PUFA are present in high concentration in grains as well as in many seeds and meats. From this reason we can notice an increase in the human consumption of seafood during several last years. Oleaginous microorganisms, as alternative sources of PUFA to others such as animal oil products, have been widely studied. Marine fungoid protists (Thraustochytrids) like Schizochytrium have been found to be as a novel, excellent DHA and EPA ω-3 fatty acids producers [24, 25]. An excellent review paper performed by Nichols demonstrates that many microorganisms including marine bacteria have been considered the major de novo producer of n-3 PUFA [26]. Available literature data suggest that over the past several years extensive research has been made for the production of PUFA by fungi [27, 28]. As it was reported by Arjuna among various microorganisms, an optimal source of omega-6 polyunsaturated fatty acids specifically γ-linolenic acid (GLA) can be certain fungi [27].

It is well known that PUFA can affect many physiological processes including cardiovascular, neurological, and immune functions, as well as cancer. Consumption of oils rich in n-3 LC-PUFA during pregnancy reduces the risk for early premature birth [12]. Studies with nonhuman primates and human newborns indicate that DHA is essential for the normal functional development of the retina and brain, particularly in premature infants [13, 29]. A new paper prepared by Kaczmarski et al. demonstrated the significant role of linoleic acid and also α-linolenic acid in some symptoms of atopic dermatitis [17]. Therefore, it could be noted that PUFA are important nutraceutical and pharmaceutical targets [22].

Until today there is a lack of knowledge about the function of LC-PUFA in mammalian tissues and cells in which they are found. However, it was stated that very long-chain polyunsaturated fatty acids are known to accumulate in two types of major genetic peroxisomal diseases, Zellweger syndrome and X-linked adrenoleukodystrophy (X-ALD), characterized by neurodegenerative phenotypes [30, 31]. The study of Hama and coworkers showed existence of many LC-PUFA types in specimens from Zellweger patients suggesting the possibility of a new biomarker for peroxisomal diseases [31]. Review paper performed by Agbaga and coworkers [32] summarized the current knowledge of VLC-PUFA to their functional role in the retina which are highly enriched in PUFA with special emphasis on the elongases responsible for their synthesis by ELOVL4 protein. Interest in LC-PUFA was rekindled in 1987 after LC-PUFA were initially detected in bovine retinas by Aveldano [33]. Effect of retinal LC-PUFA on rod and cone photoreceptors was also described by Bennett et al. [34]. Another paper prepared by Butovich confirmed that, among different mammalian tissues, meibum is an exceptionally complex mixture of various fatty acids [35].

Because most of LC-PUFA including omega-3 and omega-6 fatty acids cannot be synthesized in enough quantity by the human organism, they must be supplied in the diet. The problem of supplementation of LC-PUFA and the exploration of a new (alternative to oil fish) source of LC-PUFA (e.g., marine microalgae) is widely described for few years in many papers. For this reason, there is a need to find an effective and rapid method for identification and quantification of newly developed long-chain mono- and polyunsaturated fatty acids in plants and seafood. A set of various tools such as chromatographic methods is also needed to complete the full structural characteristic of PUFA in mammalian samples, for example, in human plasma, brain, retina, or meibum. This is important for the purpose of clinical diagnosis.

Hence, this review presents various chromatographic systems, TLC, HPLC, GC, and also SFC, suitable for preseparation and accurate quantification of long-chain mono- and polyunsaturated fatty acids from different samples with emphasis on selected literature which was published during last decade.

2. Thin-Layer Chromatography (TLC) of Long-Chain Mono- and Polyunsaturated Fatty Acids

Saturated and unsaturated long-chain fatty acids (MUFA and PUFA) are basic structural elements of lipids. Therefore, chromatographic determination of fatty acids composition by TLC including LC-MUFA and LC-PUFA content is mandatory for lipids analysis in food, agricultural, and also in biological samples [36–39]. Moreover, the mono- and polyunsaturated fatty acids can be chromatographically determined by TLC in lipids from aquatic organisms (e.g., marine and freshwater fishes, shell fishes, and marine algae) [40].

It is well known that thin-layer chromatography is a classical method of separation, identification, and quantification of fatty acids [41]. The literature survey from the last decade dedicated to lipids analysis indicates that, among different analytical methods, thin-layer chromatography (TLC) and its modern version high performance thin-layer chromatography (HPTLC) are still very important tool in lipids and also in fatty acids analysis. As it was reported by Fuchs et al. [41], there are many advantages which make TLC very competitive with HPLC (high performance liquid chromatography) in the fatty acids field such as simplicity of use, less expensive cost, small consumption of solvents in comparison with HPLC, availability of analysis of several samples in parallel, and possibility of easy visualization of unsaturated fatty acids after TLC fractionation by use of suitable dyes [41]. Sherma’s report regarding TLC analysis in food and agriculture samples confirmed that quantitative HPTLC equipped with densitometer can produce results comparable to those obtained by the use of gas chromatography (GC) or high performance liquid chromatography (HPLC) [42].

Modern topic in TLC analysis of mono- and polyunsaturated fatty acids is the use of high performance thin-layer chromatography in combination with mass spectrometric detection, for example, HPTLC-MALDI-TOF/MS [43–48]. Research studies indicate that MS detection is a powerful tool for the identification of TLC spots in more detail in comparison with traditional staining methods [41].

Many types of stationary phases classified as normal (NP) and reversed (RP) are used for TLC analysis of fatty acids (MUFA and PUFA). The most popular NP layers are silica gel, alumina, cellulose, starch, polyamides, and kieselguhr [42]. Of all above-mentioned stationary phases, the best is silica gel, which can be additionally modified by impregnation with different agents. Reversed-phase TLC is usually performed on chemically bonded RP-18, RP-2, or RP-8 layers [42].

Numerous research papers by Nikolova-Damyanova and other authors showed that the main reason which explains why TLC plays a significant role in fatty acid analysis is availability of various commercial and home-made adsorbents including impregnated TLC plates [36, 41, 49–51]. Impregnation of TLC plates with a proper reagent can improve the resolution of different classes of organic compounds including fatty acids. Among different modifications of stationary phases used in TLC, impregnated silica gel is a very suitable adsorbent for MUFA and PUFA analysis [41].

2.1. Separation of MUFA and PUFA with the Use of Impregnated TLC Plates

One of the primarily used TLC impregnating procedure to separate fatty acids in complex lipid samples was silver ion TLC (Ag-TLC). Detailed information on Ag-TLC of saturated and unsaturated fatty acids was performed in several papers and books [36, 49–51]. Silver ion chromatography is based on the ability of Ag + to form weak reversible charge transfer complexes with

electrons of the double bonds of unsaturated fatty acids [36, 52]. The retention of long-chain unsaturated fatty acids depends on the strength of complexation with Ag (I), on the number of double bonds and their configuration, and also on the distance between double bonds. Literature data indicate that both home-made and precoated glass plates are used in Ag-TLC [51]. General procedures of preparation of the stationary phases for silver ion chromatographic techniques have been surveyed by Momchilova and Nikolova-Damyanova, in 2003 [53]. Among various available TLC adsorbents, silica gel is the main supporting material [53]. Impregnation of thin layer is performed by spraying or immersing the plate in solution of silver nitrate at concentration of 0.5–20% (in the case of immersion), while for the spraying procedure 10–40% solution of silver nitrate is recommended [51, 53]. The mobile phase used in argentation TLC usually consists of two or three components, for example, hexane, petroleum ether, benzene, and toluene. Moreover, small amount of acetone, diethyl ether, ethanol, methanol, or acetic acid may be added to these mobile phases [40]. Of various visualizing agents of spots, those which are most popular for Ag-TLC of fatty acids are 50% ethanol solution of sulfuric acid, phosphomolybdic acid, and a mixture of copper-acetate-phosphoric acid. Another visualizing method is spraying the plates with fluorescent indicator by 2′,7′-dichlorofluorescein in ethanol and next viewing the spots under UV light [36]. General migration rules in Ag-TLC analysis of fatty acids were described by Nikolova-Damyanova and coworkers [53, 54]. According to Nikolova-Damyanova suggestions the retention of fatty acids with more than one double bond depends on the distance between the bonds and the elution order is as follows: separated double bonds fatty acids > interrupted double bonds > conjugated double bonds longer chain unsaturated fatty acids (LC-PUFA) are held less strongly than shorter chain fatty acids and fatty acids with trans double bonds are held less strongly than fatty acids with cis double bonds [53, 54].

One of the first Ag-TLC analyses of fatty acids was performed by Wilson and Sargent in 1992 to separate PUFA having physiological interest. PUFA’s methyl esters were separated on silica gel 60 TLC plates impregnated with AgNO3. The plates were developed with toluene-acetonitrile (97 : 3, v/v). Visualization of the spots was made by the use of 3% copper acetate-8% orthophosphoric acid. It was stated that this technique is particularly useful for metabolic studies of the chain elongated PUFA [55]. In another work Wilson and Sargent showed that silver nitrate-impregnated TLC plates were helpful in separation of monounsaturated fatty acids (as methyl esters) from polyunsaturated and also from saturated fatty acids, respectively, in metabolic studies of fatty acids by human skin fibroblasts [56]. Next work by Lin et al. indicated that silica gel and hexane-chloroform-diethyl ether-acetic acid (80 : 10 : 10 : 1, v/v/v/v) were good in analysis of docosahexaenoic acid (22 : 6 DHA) in the spermatozoa of monkeys [57]. Individual phospholipids from this sample were separated by another system such as chloroform-methanol-petroleum ether-acetic acid-boric acid (40 : 20 : 30 : 10 : 1.8, v/v/v/v/v). Other literature data confirmed that argentate silica gel chromatography enabled obtaining the high purity eicosapentaenoic acid extracted from microalgae and fish oils [58]. The recent literature reviews which were focused on TLC chromatography show that, among different chromatographic materials, Ag-TCM-TLC (silver-thiolate silica gel) is very stable (in comparison with highly light sensitive Ag-TLC plates) for TLC analysis of unsaturated organic compounds including MUFA and PUFA. Dillon et al. [59] confirmed that Ag-TCM-TLC system operates similar to Ag-TLC by separating fatty acids on the degree of unsaturation (number of double bonds). The results of this analysis are comparable to those obtained by Ag-TLC. Ag-TCM-TLC method was used to analyze some polyhydrocarbons and also methyl esters of unsaturated fatty acids containing from 0 to 6 double bonds in form of methyl esters. A mixture consisting of hexane-ethyl acetate (9 : 1, v/v) was used as mobile phase. Under these conditions complete separation of fatty acids with 0–5 double bonds was observed. Resolution of fatty acids consisting of 6 double bonds from others was not achieved in this case [59].

The results presented in this section show that various commercial silica gel plates were used in separation of unsaturated fatty acids, but some of them are not suitable for derivatization process by methylation of fatty acids on TLC plates and next their quantification by gas chromatography, because it causes the loss of separated fatty acids [60]. Methylation procedure of fatty acids after their previous fractionation on argentate silica gel was used in the analyses of unsaturated fatty acids from lipid-rich seeds. In this case a mixture of hexane-diethyl ether-acetic acid and 70 : 30 : 1 (v/v/v) was used as a mobile phase. The plates were sprayed with 2′,7′-dichlorofluorescein ethanolic solution and next identified under UV lamp (at 365 nm) [60]. The impact of Ag-silica gel on TLC analysis of methylated fatty acids, for example, c9,t11-CLA (isomer of linoleic acid) in human plasma as a prior step before GC quantification was showed by Shahin et al. [61]. Another paper prepared by Kramer et al. demonstrated that the best technique to analyze the CLA and trans 18 : 1 isomers in synthetic and animal products is the combination of gas chromatography with Ag-TLC or with Ag-HPLC [62]. Moreover, usage of Ag-TLC in the separation of isomeric forms of EPA and DHA obtained after chemical isomerization of them (during fish oil deodorization) may be found in a paper by Fournier et al. [63].

A new application of Ag-TLC is bioanalysis. A simple and rapid TLC method for analysis of PUFA levels in human blood was developed by Bailey-Hall et al. [64].

It should be pointed out that, besides the modification of silica gel with Ag + ions, the following metal salts, Cu(I), Cu(II), Co(III), and Zn(II), can be used for impregnation of TLC plates [41, 65]. Another type of impregnating agent for TLC analysis of fatty acids (MUFA and PUFA) is boric acid. It was stated that the metabolites of arachidonic acid were satisfactorily separated on silica gel impregnated with boric acid as complexing agent and by mobile phase: hexane-diethyl ether (60 : 40, v/v) [41, 65]. Next modification of stationary phase which has impact on resolution effect of fatty acids and their derivatives such as metabolites (e.g., phospholipids) is EDTA and mobile phase containing chloroform-methanol-acetic acid water in volume composition of 75 : 45 : 3 : 1 [41, 66]. Another work showed that efficient separation of five different phospholipids could be achieved by impregnation of TLC plates with 0.4% ammonium sulfate. A mixture of chloroform-methanol-acetic acid-acetone-water in volume composition of 40 : 25 : 7 : 4 : 2 was suitable for this procedure [67].

Besides the above-presented TLC system in normal phase (NP-TLC), unsaturated fatty acids and their metabolites could be separated on RP-TLC plates. One of the first reports which are focused on RP-TLC analysis of PUFA was made by Beneytout and coworkers in 1992 [68]. Beneytout et al. separated arachidonic acid and its metabolites on reversed-phase layer. The plates were silica gel coated with phenylmethylvinylchlorosilane. A mixture of heptane-methyl formate-diethyl ether-acetic acid (65 : 25 : 10 : 2, v/v/v/v) was applied as mobile phase [68].

2.2. 2D-TLC of MUFA and PUFA

Two dimensional TLC (2D-TLC) is one of the newly developed powerful tools to separate various lipids mixture and fatty acids coming from lipids. It is known that 2D-TLC improved the quality of separation, but it is much more time consuming in comparison with very popular 1D-TLC [41]. Literature review showed that 2D-TLC is rather a method of choice for separation of lipids from cell membrane polyphosphoinositides and also of lipid oxidation products in mixture. This analysis is usually performed on silica gel impregnated with magnesium acetate (7.5%) and by solvent system chloroform-methanol-ammonia (5 : 25 : 5, v/v/v) in the first direction and chloroform-acetone-methanol-acetic acid water (6 : 8 : 2 : 2.1, v/v/v/v) in the second dimension [69].

2.3. Detection of Spots and Quantification Methods of MUFA and PUFA

Detection of fatty acids by TLC method is based on their visualization by binding to a dye. As is was reported in excellent review by Fuchs et al. [41] a lot of visualizing reagents suitable for detection of fatty acids are described in the literature. Among them, the most popular reagents are iodine vapors, 2′,7′-dichlorofluorescein, rhodamine 6G, which produce coloured spots, and also primuline, which gives sensitivities in the nanomole range [41]. In case of PUFA intense darkening is achieved after their separation on AgNO3 impregnated TLC plates (as an effect of reduction of Ag + to colloid silver), but this method of detection required the presence of aromatic hydrocarbon as mobile phase component [70]. Other visualizing reagents are as the following: sulfuric acid, potassium dichromate in 40% sulfuric acid, or 3–6% solution of cupric acetate in phosphoric acid. Moreover, detection of different fatty acids is possible by PMA (phosphomolybdic acid) and by sulfuryl chloride vapors [41]. Visualization of fatty acid spots is performed by spraying or dipping the plates in solution of respective visualizing agents. Next, the spots are observed under UV light or identified by densitometry. For more detailed characterization of fatty acids which have been separated by thin-layer chromatography, TLC combined with mass spectrometer (TLC-MS) may be used. In this method the spots are eluted from the chromatographic plates with respective solvents and next obtained fatty acids are analyzed by MS. Applying of TLC coupled with MS enables high resolution of identified peaks. Moreover, there is no need to extract sample from the plates prior to this analysis [41]. A novelty in thin-layer chromatographic instrumentation is a TLC in combination with MALDI MS spectrometer (TLC MALDI) [44, 71, 72]. This technique is rather fast and provides spectra that can be relatively simply analyzed and tolerates high sample contamination [41]. The detection limit of fatty acids determined by TLC MALDI might be less than 1 nanogram [41]. It was stated that TLC MALDI could be satisfactorily applied to very complex lipid mixture (e.g., extracts from stem cells) [47]. For example, by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus were performed [45]. Next modern trend in analysis of lipid profile is the use of TLC method coupled with FID (flame ionization detector) [73]. Chromarod/Iatroscan TLC-FID was successfully used in the analysis of lipid classes and their constituents of fatty acids extracted from seafood. As it was described in the paper by Sinanoglou et al. [73], Iatroscan is an instrument that combines TLC resolution with capacity of quantification by FID. Efficient TLC-FID separation can be achieved by addition of polar solvent system without changing the stationary phase. However, this apparatus allows analyzing in a short time (2-3 hr) in comparison with GC or HPLC about 30 samples [73].

2.4. TLC Separation of cis and trans Isomers of MUFA and PUFA

Since it was reported that the saturated fatty acids indicate correlation with cardiovascular diseases, unsaturated fatty acids have been recommended for replacement of saturated fatty acids in a diet. For this reason, an increase of interest in unsaturated fatty acids such as n-6 and n-3 fatty acids is observed. It is known that the discussed unsaturated fatty acids form specific geometrical isomers. They can be trans or cis depending on the orientation of double bond. Of all polyunsaturated fatty acids the trans PUFA consisting of C18, C20, and C22 chain lengths are usually part of the human diet. Thus, it is very important to detect and quantify them in food products. One of the most popular method obtaining the mono-, di-, and triunsaturated fatty acids in form of geometrical isomers (cis and trans), respectively, is thermal or chemical process [63]. Synthesis of trans isomers is usually made by food manufacturing (refining, hydrogenation). For instance, trans isomers are formed during deodorization (crucial step of refining) of vegetable or fish oils. As it was reported by Fournier et al. [63], the methodologies regarding accurate separation and quantification of trans isomers of mono-, di-, and triunsaturated fatty acids by chromatographic methods were developed in the last decade. Ag-TLC is one of the most powerful chromatographic techniques widely applied to separate cis and trans isomers of LC-PUFA because it is characterized by simplicity, low cost, and efficiency. Geometrical isomers are separated according to their number of double bonds. The efficacy of Ag-TLC for separation of EPA and DHA isomers was confirmed by Fournier et al., in 2006 [63]. For this purpose TLC plates precoated with silica gel and impregnated by AgNO3 were used. A mixture of toluene-methanol in the volume composition 85 : 15 was applied as a mobile for resolution of EPA mono-, di-, tri-, tetra-, penta-trans and DHA hexa-trans isomers [63]. In another work, in order to determine the profile of cis and trans isomers of CLA in liver by the TLC method, a mixture of trichloromethane-n-hexane-glacial ethanoic acid 65 : 35 : 1 (v/v/v) and also silica gel plates were applied. Rhodamine solution was used as a visualizing agent. In further steps, after methylation of separated fatty acids they were quantified by GC method [74].

Next paper indicated that preparative silver nitrate thin-layer chromatography was successfully applied to analyze the cis,cis-octadecadienoic acid (18 : 2) in commercial samples of bovine butter fat. The detected by Ag-TLC cis,cis-5,9-18 : 2 isomer was found for the first time in butter fat [54]. In another food study, ninety-three commercial samples of Bulgarian butter fats manufactured evenly through the year were subjected to quantitative silver nitrate-TLC of fatty acids components (as isopropyl esters), with particular attention to trans monoenoic fatty acid content [75]. All performed results indicate that argentation TLC should be an effective analytical technique in fractionation and identification of geometric isomers of unsaturated fatty acids such as PUFA.

Table 1 shows the most efficient TLC systems used for separation and fractionation of fatty acids from various matrices.

3. High Performance Liquid Chromatography (HPLC)

There are few excellent original papers and reviews until today which are focused on usage of liquid column chromatography for the analysis of fatty acids (saturated and unsaturated) and also their related substances in biological, food, and drugs samples [18, 93–96]. In these papers principles of HPLC including the sample preparation, mobile phases, stationary phases, and detection methods were widely performed. Among the earlier review papers, only three of them survey knowledge of long-chain fatty acids. One of them prepared by Rao et al. [94] showed the LC-PUFA analysis with use of HPLC but since 1974 until 1995. Next paper supported by Rezanka and Votruba [96] demonstrated the use of chromatography including HPLC for analysis of very long-chain fatty acids from 1982 to 2001. The third review prepared by Kolanowski and coworkers [97] described the important instrumental methods such as HPLC and also GC for analysis of omega-3-long-chain polyunsaturated fatty acids but in food only. Lack of the overview about the new achievements in HPLC analysis of mono- and polyunsaturated fatty acids with an emphasis on the analysis of long-chain polyunsaturated fatty acids such as omega-3 and omega-6 causes that there is a necessity to perform a complete literature survey from 2002 to 2013 (last decade) with emphasis on application of HPLC for the analysis of all biological important LC-MUFA and LC-PUFA at the analytical levels in various matrices. This paper highlights the modern achievements of the HPLC including the principles of MUFA and PUFA analysis in different matrices: separation modes and the new detection systems which enable quantification of LC-PUFA at nanogram level. The current knowledge of HPLC analysis of mono- and polyunsaturated fatty acid was described on the basis of the works which were published during the past decade (2002–2013).

The use of HPLC for the separation and quantification of fatty acids increased since 1950, when it was applied for first time by Haward and Martin for analysis of some fatty acids [98]. From this time until today a very quick progress in HPLC analysis of all fatty acids including LC-MUFA and LC-PUFA is observed. It is known that, based on nature of stationary phase (solid or liquid), the high performance liquid chromatography is divided into [18] liquid-liquid chromatography (LLC), adsorption chromatography (LSC), and reversed-phase chromatography (RP), which is a combination of LSC and LLC.

Among above-mentioned high performance liquid chromatographic techniques RP-HPLC plays a key role in LC-PUFA analysis [94]. This technique allows separating those fatty acids which cannot be separated by normal phase HPLC. As it was early reported by Rao et al. [94] the retention of fatty acids in RP-HPLC depends on the polarity of the stationary phase, mobile phase, and chemical structure of examined fatty acids. In general, the retention time is proportional to the chain length and the number of double bonds present in examined fatty acids. Additionally, the influence of geometric isomerization of fatty acids plays a very important role in reversed-phase HPLC analysis. It was reported that the cis isomers of the fatty acids are generally eluted before trans isomers. Similar effect is observed in case of the differences in number of carbons in studied fatty acids. Those with small amount of carbons (short chain) are eluted first in comparison with long-chain fatty acids [94]. The current literature indicates that the reversed-phase HPLC is one of the popular methods used in the field of fatty acids analysis of MUFA and PUFA because its simplicity, reproducibility, and credibility. The results obtained by HPLC are usually comparable with those determined using GC method. Moreover, the time of analysis is comparable to GC technique [94]. However, development of new detector mode such as flame ionization detector (FID) or electrochemical detector (ED) enhances the applicability of HPLC for MUFA and PUFA analysis [93].

3.1. Derivatization Methods of Fatty Acids for HPLC Analysis

The most important problem in HPLC study of LC-MUFA and also LC-PUFA is a need to detect them at lower concentration levels such as nanograms or picograms. Derivatization of fatty acids can improve the importance for HPLC analysis parameters such as sensitivity, precision, selectivity, and also a limit of detection and quantification [18, 95]. Different derivatization methods used for quantitative and qualitative determination of fatty acids were widely described in review paper by Rosenfeld in 2002 [99]. Generally, the kind of derivatizing agent depends on the type of detector applied in PUFA analysis including UV absorption, fluorescence, light scattering, and refractive index detectors [94].

UV-VIS detector is the most popular detection system applied in liquid chromatography because it is sensitive and specific. In 1983 Aveldano and coworkers [100] have applied reversed-phase high pressure liquid chromatography with UV detection on octadecylsilyl column to separate a mixture of underivatized unsaturated and saturated fatty acids and their methyl esters coming from mammalian tissues.

Generally in order to facilitate the detection by UV-VIS absorption the following derivatizing reagents for fatty acids are used [93]: phenacyl bromide (PB), p-bromophenacyl bromide (BPB), p-chlorophenacyl bromide (CPB), p-nitrophenacyl bromide which are used in analysis of fatty acids in oils, standards, and blood in the range from ng to pmol [97]. Naphthyl esters (obtained by 2-naphthyl bromide, p-nitrophenacyl, and p-dichlorophenacyl allow for the detection of picograms fatty acids in standard mixture. 2-Nitrophenylhydrazine (NPH) and 2-bromoacetophenone (or α,p-dibromoacetophenone, BAP) convert the fatty acids into specific derivatives which can be detected in biological samples (serum) with detection limits in fmoles. PNB (p-nitrobenzyl) and p-methylthiobenzyl chloride (MTBC) derivatives of fatty acids with detection limits in pmoles are important in coconut oil analysis. Ideal derivatizing reagent for chiral separation of fatty acids is 3,5-dinitrophenyl isocyanate (DNPI) [96].

Next very popular detection system applied in HPLC is fluorescence detector which has higher sensitivity in comparison with UV-VIS detector. For this reason it could quantify the fatty acids composition at picomole and femtomole level. Several fluorescent reagents are used for the derivatization of fatty acids such as 9-diazomethylanthracene (9-DMA) and 9-anthryldiazomethane (ADAM). Anthryl methyl esters of fatty acids can be easily detected in human plasma and serum in picomoles. Other more sensitive derivatives than ADAM are the esters of pyrenyldiazomethane (PDAM). Coumarins and 9-aminophenanthrene (9-AP) are important in detection of fatty acids in environmental and in biological samples in the range from pmoles to fmoles. Other derivatives like dansyl piperazines and acetamides of fatty acids can be detected in serum below 100 fmol range [93, 94, 97]. Recent study performed by Wang and coworkers in 2013 indicates that using a new fluorescent labeling reagent named 1,3,5,7-tetramethyl-8-butyrethylendiamine-difluoroboradiaza-s-indacene (TMBB-EDAN) a sensitive and rapid HPLC technique for the determination of fatty acids in biosamples (e.g., human serum) was developed. With the proposed procedure the limit of detection of fatty acid derivatives was in the 0.2–0.4 nM range [101].

Another type of fatty acids detection is chemiluminescence. This method is based on the prelabeling of the –COOH groups with a proper chemiluminogenic reagent. In practice luminal and its related compound isoluminal have been widely utilized as chemiluminescence derivatization reagents for fatty acids because of their chemiluminescent properties. Described detection system is efficient for the sensitive detection of fatty acids in human serum and plasma in picomoles [93].

In order to detect the substances with oxidizing or reducing properties including fatty acids an electrochemical detector (ED) may be used. This detection mode enables measure of fatty acids in complex biological samples containing different components at nanogram levels. To obtain the higher sensitivity the following derivatizing reagents for ED can be applied: p-aminophenol 2,4-dimethoxyaniline 2-bromo-2′-nitroacetophenone ferrocene derivatives 2,4-dinitrophenylhydrazine and 3,5-dinitrobenzoyl chloride. Clinical application of HPLC-ED is showed in the paper by Kotani et al. [80]. In this work determination of plasma fatty acids including PUFA such as arachidonic acid and also linoleic by high performance liquid chromatography coupled with electrochemical detector (HPLC-ED) was performed [80]. This procedure may be found suitable for monitoring plasma fatty acids in diabetic patients.

Among different types of detectors used in HPLC analysis of fatty acids, very useful is that detector which requires none of the preliminary derivatization procedures of studied compounds like, for example, evaporative light-scattering detector (ELSD) or mass spectrometer (MS) [93]. As it was described in paper by Lima and Abdalla [93] evaporative light-scattering detector is sensitive to mass of vaporized analyte and its operation system is not limited by the absorption characteristics of the individual components and nature of the eluent. Thus, ELSD may be used to solve the problem of separation of fatty acids which have weak absorbance groups [93].

3.2. Mass Spectrometry Detection of Fatty Acids

Application of HPLC-MS in analysis of fatty acids is known only from the last decade. Mass spectrometer is extensively used in analysis of fatty acids especially in case of biological samples. One of the main advantages of HPLC-MS method is possibility to analyze nonvolatile compounds including fatty acids [93]. Numerous ionization and detection modes can be applied for fatty acids analysis such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and also time of flight (TOF). A new possibility in HPLC-mass spectrometry is tandem MS (HPLC-MS-MS) or combination of electrospray ionization with tandem mass spectrometer such as HPLC-ESI-MS-MS technique. The major advantage of LC-ESI is the great separation of fatty acids in complex matrices like blood plasma. HPLC-MS-MS is a powerful tool in determination of double bond position or branched points in the chain of unsaturated fatty acids [93]. Next HPLC method developed for detection of long-chain fatty acids is HPLC-MS method combined with APCI, which was applied in detection of very long-chain fatty acids in form of picolinyl esters coming from sugar cane wax [102]. HPLC-MS coupled with three ionization systems, electron impact (EI), atmospheric pressure chemical ionization, and electrospray ionization, was suitable for the accurate determination of PUFA and also their oxidative metabolites such as eicosanoids in biological samples in the range of pg [103]. According to papers prepared by Řezanka et al., which were focused on fatty acids analysis in lower organisms, liquid chromatography mass spectrometry coupled with ionization mode APCI was the most efficient method applied for identification and quantification of very long-chain polyunsaturated fatty acids from marine organisms (which have ability to produce VLC-PUFA) such as marine dinoflagellates Amphidinium carterae and green algae Chlorella kessleri [81, 104–106] and also in oil obtained from Ximenia fruits (raw material for cosmetic industry) [107]. As it was performed in these papers HPLCMS-APCI system consisted of Hichrom column (HIRPB-250AM) and gradient solvent program with acetonitrile (MeCN), dichloromethane (DCM), and propionitrile (EtCN) which were suitable for separation 13 of picolinyl esters of VLCPUFA from lower organisms. Excellent separation of methyl esters of unsaturated fatty acids coming from freshwater crustacean was achieved by the same mobile phase and the chromatographic column packed with octadecylsilyl phase (Supelcosil LC-18) [108]. An extensive review of a very long polyunsaturated published by Řezanka and Sigler indicates that the modern analytical methods such as HPLC-MS make the detection and identification of VLC-PUFA possible in different classes of lipids including microbial kingdoms and fungi [109]. Reversed-phase HPLC with gradient elution of solvent system containing acetonitrile and chloroform and equipped with light-scattering detector (ELSD) was used to purify and identify the methyl esters of C16–C28 PUFA including octacosaoctaenoic acid in milligram quantities from marine microalgae [82]. A simple method based on reversed-phase ion-pair high performance liquid chromatography (RP-HPLC) was used successfully to separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic, and docosahexaenoic acids [110]. These compounds were easily identified by liquid chromatography mass spectrometry (HPLC-MS) with atmospheric pressure chemical ionization (APCI) in nanogram range [110]. This work demonstrated that the method based on APCI-MS coupled with HPLC is highly reliable for the analysis of various monoepoxides of PUFA in their metabolism study. Another HPLC system such as nonaqueous reversed-phase high performance liquid chromatography (NARP-HPLC) with atmospheric pressure chemical ionization (APCI-MS) was suitable for detection of 5 unsaturated polymethylene interrupted fatty acids such as cis-5,9-octadecadienoic (taxoleic), cis-5,9,12-octadecatrienoic (pinolenic), cis-5,11-eicosadienoic (keteleeronic), and cis-5,11,14-eicosatrienoic acids (sciadonic) isolated from conifer seed oils (obtained from European Larch, Norway Spruce, and European Silver Fir) [111]. Combination of two chromatographic methods such as TLC and HPLC was used to analyze n-3 fatty acids in fish oil dietary supplements. The EPA and DHA fraction obtained by means of Ag-TLC method were further analyzed by HPLC. The chromatographic column ODS (3.9 mm × 30 cm, 10 μm), mobile phase containing tetrahydrofuran-acetonitrile-water-acetic acid 25 : 35 : 75 : 0.4 (v/v/v/v), and photodiode array detector in the range of 190–240 nm were used in this analysis [112]. HPLC system equipped with analytical column Supelcosil C18 (4.6 mm × 25 cm) and photodiode array detector (DAD) was used for the analysis of hydroperoxy PUFA as the products of peroxidation coming from human plasma. A mixture of acetic acid-acetonitrile-tetrahydrofuran 52 : 30 : 18 (v/v/v) as mobile phase was used. The analysis was monitored at 200–300 nm. Quantity control by applied procedure of obtained derivatives of PUFA may be useful as clinical markers of oxidative stress on biological systems [113]. In another paper gradient reversed-phase liquid chromatography (C18 ODS 25 cm × 0.46 cm, 5 μm) by use of methanol water and universal ELSD detector allowed for separation and fractionation of saturated, unsaturated, and oxygenated free fatty acids as methyl esters extracted from seeds of Crepis alpina and Vernonia anthelmintica, respectively [114]. This work showed that reversed-phase C18 was suitable for purification and fractionation of PUFA before their quantification by GC-MS. Besides HPLC system equipped with above-mentioned types of detector modes such as ECD, MS, ELSD, DAD, and UV, a fluorescence detector is also very useful [115, 116] which enabled precise determination of long-chain polyunsaturated fatty acids including linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids in human serum at low concentrations like fmoles. For example, the method described in 1986 by Yamaguchi et al. performed the conversion of LC-PUFA into fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone. Separation process was performed on a reversed-phase column (YMC Pack C8) with an isocratic elution using aqueous 72% (v/v) acetonitrile [115]. Current literature shows that HPLC with different solvent systems is suitable in analysis of novel hydroxyl fatty and phosphoric acid esters of 10-hydroxy-2-decenoic acid (9-HAD) coming from the royal jelly of honeybees (Apis mellifera). HPLC analysis was carried out on Diaion HP-20, Sephadex LH-20, and Cosmosil 75C18-OP column systems. Different mobile phases containing acetonitrile were applied. A refractive index detector was applied to monitor the elution profile of examined fatty acids [117].

3.3. Silver Ion HPLC in Quantification and Identification of Geometric Isomers of Fatty Acids

As it was reported by Nikolova-Damyanova and Momchilova [118], silver ion HPLC (Ag-HPLC) is widely applied by many scientists as a preliminary step for the fractionation of complex mixture of fatty acids into their groups prior to quantitative analysis by GC method. Resolution of fatty acids by means of Ag-HPLC is achieved according to the number and geometry of double bonds [118]. Separation is based on the reversible formation of a weak charge-transfer complex between a silver ion and a double bond. The main problem in Ag-HPLC is introduction of Ag + into this system. Thus, similar to the case of Ag-TLC the first attempt was performed on column which was laboratory packed with slurry of the stationary phase (e.g., silica gel) impregnated with AgNO3. Second method is to add silver ion solution (AgNO3) into mobile phase. Third method is to use commercially available silica based cation exchange column (Nucleosil 5 SA), or the column which is produced by Chrompack (ChromSpher 5 lipids). These commercial column systems give much better results (better reproducibility) of fatty acids analysis than those laboratory prepared [118]. Another excellent review of the chromatographic methods used to analyze geometrical and positional isomers of fatty acids by Aini et al. [119] showed that the following factors affect resolution of fatty acids in silver ion HPLC such as the impregnation method of column, mobile phase composition, and also column temperature [119]. The choice of mobile phase composition used in Ag-HPLC is usually toluene based, acetonitrile based, hexane based, or dichloromethane based [118, 120]. Good results have been achieved by the use of isopropanol and tetrahydrofurane as a modifier. The lower column temperature generally results in shorter elution time in Ag-HPLC. Moreover, the improvement in resolution of monoenoic and polyenoic fatty acids was obtained by converting them into phenacyl, benzyl, n-propyl, n-butyl, and ethyl isopropyl esters [52]. Separation by Ag-HPLC coupled with UV detector of cis- and trans-octadecanoic acids described by Momchilova and Nikolova-Damyanova [83] indicated that the efficiency of the separation increases in the following order: phenethyl < phenacyl < p-methoxyphenacyl esters. Yet, retention and resolution of these fatty acids by Ag-HPLC could be affected by small changes of dichloromethane in mobile phase. Among various chromatographic systems the best resolution of cis- and trans-positional isomers of octadecenoic acid after converting them into p-methoxyphenacyl esters was achieved on a silver ion column by isocratic elution with a mobile phase containing hexane-dichloromethane-acetonitrile in volume composition of 60 : 40 : 0.2 (v/v/v) [120]. Similar mobile phase was used by Momchilova and Nikolova-Damyanova in 2000 to estimate the chromatographic properties of positional isomers of octadecenoic fatty acids after conversing them to 2-naphthyl, 2-naphthylmethyl, and 9-anthrylmethyl derivatives [83]. According to the results obtained in this paper, dichloromethane-acetonitrile 100 : 0.025 (v/v) as mobile phase provided better resolution of 9-anthrylmethyl derivatives of 6-, 9-, and 11–18 : 1. High resolution by Ag-HPLC was obtained for the positional isomers of PUFA such as eicosapentaenoic acid, docosahexaenoic acid, and also docosapentaenoic acid coming from triacylglycerols containing PUFA. The hexane-isopropanol-acetonitrile solvent system was suitable for the separation of these compounds [84]. Isolation of some PUFA from edible oils by argentate silica gel chromatography (Ag-TLC and Ag-HPLC) was performed by Guil-Guerrero et al. [121]. Using this method the isomers of the following fatty acids, linoleic acid, α-linolenic, γ-linolenic, and stearidonic acid, and eicosapentaenoic and docosahexaenoic acid, have been isolated in form of methyl esters from linseed, sunflower, and borage seed oils and from shortfin mako liver oil. Similarly, like it was described in the previous part, Ag-HPLC was useful for analysis of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization [63]. Obtained results showed that this technique cannot be used to determine the isomers in fish oil which have been formed at the temperature higher than 180°C (e.g., at 220°C), because the interference between obtained isomers, especially di-trans DHA and all-cis EPA, was observed. Efficient analysis of trans isomers of conjugated linoleic acid in synthetic and animal products (from pigs, chicken meat) was achieved also by chromatographic techniques including Ag-HPLC [62]. Recent literature indicates that quantification of separated by Ag-HPLC geometric isomers of fatty acids is mainly performed by GC-MS or by GC coupled with FID (flame-ionization detector). FID, UV, and refractive detector are usually used for direct quantification of isomers of fatty acids as methyl esters. As it was reported by Nikolova-Damyanova and Momchilova [118] aromatic esters of fatty acids can be detected by Ag-HPLC combined with UV-VIS detector in the range of 0–200 μg. The paper prepared in 2013 by Sun and coworkers demonstrates that a liquid chromatography/in-line ozonolysis/mass spectrometry (LC/O3-MS) is a practical and easy to use new approach to direct determination of double bond position in lipids presented in complex mixture. In order to test this method in complex lipid extracts, a sample of bovine fat with known amount of positional isomers and cis/trans isomers of unsaturated fatty acids was analyzed. The main advantage of the in-line ozonolysis is its applicability in combination with various mass spectrometers without instrumental modification [122].

Survey of some selected HPLC conditions useful for separation and identification of MUFA and PUFA from various samples is listed in Table 2.

Watch the video: Reversed phase liquid chromatography (February 2023).