U.S. Reporter 29.5

Liquid Chromatography & HPLC, Supelco Reporter 29.5

Serum Lipidomics Profiling using an Ascentis Express Fused-Core C18 Column
HPLC Method for the Separation of Nine Key Components of Milk Thistle

Serum Lipidomics Profiling using an Ascentis Express Fused-Core C18 Column back to top

  The following was generated with the assistance of an outside source using Sigma-Aldrich products. Technical content was generated and provided by:
Susan S. Bird and Bruce S. Kristal
Department of Neurosurgery, Brigham and Women’s Hospital and, Department of Surgery, Harvard Medical School, 221 Longwood Avenue, LMRC-322, Boston, Massachusetts 02115
Lipid diversity in a given biological system is quite great as there are both charged and neutral lipids in addition to species of varying degrees of chemical polarity. The LIPID Metabolites And Pathways Strategy (LIPID MAPS) consortium (www. lipidmaps.org) has systematically defined and classified lipids into eight categories, with multiple subclasses of each covering both eukaryotic and prokaryotic sources (Figure 1) (1). This standardization has assisted the progress in developing analytical methods for untargeted lipidomic profiling studies where all lipids are of potential importance and therefore require sensitive and robust detection.
Figure 1. The Inherent Diversity of Lipids and the Systematic Classification of Each Lipid Type by the LIPID MAPS Consortium

Chromatographic separation of lipid extracts prior to mass spectrometric (MS) detection aids in the achievement of comprehensive, quantitative, and reproducible analytical data in an efficient and robust manner. In the past, successful separation and analysis of lipids has been achieved using both gas chromatography (GC) and normal phase LC coupled to MS. These techniques, however, are not without limitations relating to what types of lipids are detectable and to the run-to-run reproducibility of the method (2, 3). Reversed phase (RP) LC is often the technique of choice due to its efficient separations and ability to interface easily with electrospray ionization-MS detection (4-7).

The current study uses a Fused-Core® C18 RP-LC column to achieve lipid separations in biological samples. The Fused-Core technology offers more robust particle size distribution, in comparison to traditional porous particles, that allow for sharper peaks by minimizing the eddy diffusion through the column. Additionally, this type of particle has a reduced diffusion path that provides sharper peaks at higher flow rates allowing for increased sample throughput (4). Lipidomic analysis of serum samples from a rat diet study (n=192) were analyzed using RP-LC-MS in order to achieve comprehensive lipid coverage, relative quantiation of lipid species between diets and characterization of unknown lipids. The methods ability to reproducibly separate and detect 86 unique triglycerides in addition to lipids from the other 6 categories found in plasma will be highlighted.

Animal Study
Sets of male Fisher 344 x Brown Norway F1 (FBNF1) rats (n=8 per set), aged 7-9 weeks, were fed ad libitum one of 24 isocaloric diets that differed in fat and carbohydrate composition (total n=192).

Lipid Extraction and LC-MS Analysis
Lipid extracts were extracted from 30 ìL of rat serum using a modified Bligh and Dyer liquid-liquid extraction and separated on an Ascentis® Express C18, 15 cm x 2.1 mm I.D., 2.7 µm column (Sigma-Aldrich, St. Louis, MO) connected to a Thermo Fisher Scientific PAL autosampler, Accela quaternary HPLC pump and an Exactive benchtop orbitrap mass spectrometer (Thermo Fisher, San Jose, CA) equipped with a heated electrospray ionization (HESI) probe. The HPLC was run at 260 µL/min and the column was held at 550 °C. Mobile phase A consisted of 60:40 acetonitrile:water in 10 mM ammonium formate with 0.1% formic acid and mobile phase B consisted of 90:10 isopropanol:acetonitrile also with 10 mM ammonium formate and 0.1% formic acid. Details of the LC-MS method and SIEVE analysis have been described previously (6, 7).

Lipid Class Separation
Because many lipid species are isobaric, an understanding of the chromatographic retention time pattern can facilitate the identification and characterization of unknowns. The Acentis Express reversed phase C18 column separated lipids by both head group polarity and acyl side chain composition in an efficient manner yielding sharp and robust peaks. Figure 2 shows representative total ion current (TIC) chromatograms of a serum sampled pooled from all 192 animals in our study. Sections of the chromatograms are labeled with the lipid categories detected, indicating the regions where each will elute using the LC-MS method. Highlighted in italics are some specific lipid classes which are found from those categories that are observed in the serum pool samples. The diverse nature of the serum lipidome suggested that comprehensive LC-MS profiling methods utilize both positive and negative ionization modes.
Figure 2. Panels A and B Show the Total Ion Chromatogram (TIC) Separation of the Same Serum Pool Sample Using LC-MS Performed in Negative and Positive Ionization Modes, Respectively

Triacylglyceride Separation
In the positive ion TIC (Figure 2) the majority of signal is due to the highly abundant serum triglycerides (TGs). There is a growing understanding that there are TG species-specific implications for health (8) therefore our priority was to identify all serum TGs found in our animal dietary macronutrient study. Identification was achieved by first analyzing a standard mixture of 6 TGs —TG (8:0)3, TG (10:0)3, TG (12:0)3, TG (14:0)3, TG (15:0)3 and TG (16:0)3— and determining the elution profile and retention time (RT) reproducibility of these species. From Figure 3, you can see that separation is based on acyl chain length. From repeat injections of these standards over 5 days we determined the RT coefficients of variation to be < 0.3%. This experiment allows us to recognize regions in the chromatogram where we can expect the TGs to be found, noting that the biologically relevant TGs will contain longer acyl side chains and be located between 22-25 minutes. 
Figure 3. Separation of 6 TG standards —TG (8:0)3, TG (10:0)3, TG (12:0)3, TG (14:0)3, TG (15:0)3 and TG (16:0)3.

Unknown Triacylglyceride Identification
When analyzing our unknown rat serum data, exact mass measurement and retention time pairs from SIEVE that fell into the predetermined regions of the chromatogram were searched through online databases for possible TG hits. All ion fragmentation MS was then used to fully characterize these hits. Extracted ion chromatograms (XIC) of the fragment and parent masses were aligned to determine the FA side chains on each TG molecule.

Figure 4 shows an example of this type of identification. A lipid with m/z 852.8015 found at RT 24.06 minutes, is found to match the mass of TG (50:0) as an [M+NH4]+ ion when searching the human metabolome database and yields two possible TG isomers. In panels B and C, the diacylglycerol fragments chromatographically align with the parent ion to confirm the structure as TG (16:0/16:0/18:0). We present the specifics on the MS studies involved in the characterization of TGs, in a recent paper in Analytical Chemistry (7).
Figure 4. The XIC of parent ion m/z 852.8015 is shown in panel A, with panels B and C showing XICs of the diacylglycerol fragments of the molecule. Chromatographic alignment of the parent ion peak at 24.05 minutes can be seen with fragments m/z 579 and m/z 551
In a profiling LC-MS method, lipid separation is very important since it is natural for co-elution to occur when a large number of species elutes over a limited time period. In addition to identifying lipids from 6 major categories found in rat serum, this profiling method was able to identify 86 unique TGs with 62 having their individual side-chains characterized. This is a significant improvement over past methods where not only were far fewer TGs identified; they were merely characterized by their total number of carbons and double bonds. This enhancement is a reflection of the added chromatographic resolution observed with the Ascentis Express Fused-Core column and the high resolution accurate mass detection of our MS system. 

  1. Fahy, E.; Subramaniam, S.; Brown, H. A.; Glass, C. K.; Merrill, A. H., Jr.; Murphy, R. C.; Raetz, C. R.; Russell, D. W.; Seyama, Y.; Shaw, W.; Shimizu, T.; Spener, F.; van Meer, G.; VanNieuwenhze, M. S.; White, S. H.; Witztum, J. L.; Dennis, E. A. J Lipid Res 2005, 46, 839-861.
  2. Sumner, L. W.; Mendes, P.; Dixon, R. A. Phytochemistry 2003, 62, 817-836.
  3. Wang, C.; Kong, H.; Guan, Y.; Yang, J.; Gu, J.; Yang, S.; Xu, G. Anal Chem 2005, 77, 4108-4116.
  4. Hu, C.; van Dommelen, J.; van der Heijden, R.; Spijksma, G.; Reijmers, T. H.; Wang, M.; Slee, E.; Lu, X.; Xu, G.; van der Greef, J.; Hankemeier, T. J Proteome Res 2008, 7, 4982-4991.
  5. Rainville, P. D.; Stumpf, C. L.; Shockcor, J. P.; Plumb, R. S.; Nicholson, J. K. J Proteome Res 2007, 6, 552-558.
  6. Bird, S. S.; Marur, V. R.; Sniatynski, M. J.; Greenberg, H. K.; Kristal, B. S. Anal Chem, 2011 83, 940-949.
  7. Bird, S. S.; Marur, V. R.; Sniatynski, M. J.; Greenberg, H. K.; Kristal, B. S. Anal Chem, 2011, In Press.
  8. Rhee, E. P.; Cheng, S.; Larson, M. G.; Walford, G. A.; Lewis, G. D.; McCabe, E.; Yang, E.; Farrell, L.; Fox, C. S.; O'Donnell, C. J.; Carr, S. A.; Vasan, R. S.; Florez, J. C.; Clish, C. B.; Wang, T. J.; Gerszten, R. E. J Clin Invest, 121, 1402-1411.

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HPLC Method for the Separation of Nine Key Components of Milk Thistle back to top
Silymarin, derived from the milk thistle plant, has been used as a natural remedy for the treatment of a number of liver diseases as well as in the protection of the liver from potential toxins. More recently, silymarin has been connected to antitumor promoting activity (1). It is therefore of interest to have available analytical methods for the analysis of silymarin components in various matrices.

The silymarin complex consists of several closely related, biologically active components. The objective of this study was to investigate several modern stationary phase chemistries for optimal selectivity toward the development of an efficient method for the analysis of silymarin. The method would optimally provide baseline resolution for nine of the known components of silymarin in less than 15 minutes.

Initial screening experiments were used to select a single combination of stationary phase and organic modifier that demonstrates the greatest potential for the separation. The information from subsequent optimization experiments was processed with simulation software to predict an optimized set of conditions. Finally the predicted conditions were confirmed using the analysis of an herbal supplement obtained from a local drug store.

The screening protocol utilized five Ascentis Express stationary phases including C18, C8, RP-Amide, Phenyl-Hexyl and pentafluorophenylpropyl (F5). Gradient elution with two distinct organic modifiers was performed. Injections of a standard containing nine of the known constituents of silymarin (Figure 1) were made under each condition.

The HPLC screening method employed gradient elution from 5% - 95% of either methanol or acetonitrile. The aqueous component of the mobile phase was 0.1 % formic acid (pH 2.6).

In order to optimize the separation, separate chromatographic analyses at two different gradient slopes and two temperatures were performed. These four runs were then processed using ACD LC-Simulator software (Toronto, ON, Canada) and a predictive model was used to develop conditions that would provide baseline resolution of all peaks in less than 15 minutes. The conditions were then confirmed using both standards and a commercially available milk thistle herbal supplement.
Figure 1. Structures of Known Biologically Active Silymarin Components

Results and Discussion
Using a simple peak counting approach, the combination the C18 stationary phase along with methanol as the organic modifier resulted in the most visible peaks with nine. A representative chromatogram is shown in Figure 2. Other combinations of stationary phase and organic modifier produced between six and eight peak responses.

Four separate chromatographic runs using the C18 stationary phase and methanol modifier were conducted where the gradient slopes were varied (5% and 10% ramp) at two different temperatures (30 °C and 60 °C). The data was then analyzed using ACD LC-Simulator to predict the most suitable optimized conditions. Comparison to the experimental data shown in Figure 3 confirms good agreement between the predicted (not shown) and actual chromatography. Because the suggested conditions ran to just 45% organic, an additional step to 100% organic was included to elute potential hydrophobic components from a natural sample.
Figure 2. Initial Separation of Silymarin Components using C18 Stationary Phase and Methanol as the Organic Modifier
Figure 3. Experimental Confirmation of Predicted Optimized Conditions
column:   Ascentis Express C18, 10 cm x 3.0 mm I.D., 2.7 µm particle size (53814-U)
Mobile Phase A:   water with 0.1% formic acid
Mobile Phase B:   methanol
min %A %B
0 65 45
3 65 45
13 55 45
15 55 45
flow rate:   0.6 mL/min
temp.:   35 °C
det.:   UV, 254 nm
injection:   20 µL
sample:   9 milk thistle related 20 ìg/ìL in 86:14, water:methanol
1. Taxifolin
2. Silychristin
3. Apigenin-7-D-glucose
4. Silydianin
5. Quercitrin
6. Silybin A
7. Silybin B
8. Isosilybin A
9. Isosilybin B

Finally an herbal supplement labeled as containing milk thistle along with dandelion, fennel and licorice was extracted using water:ethanol 50:50, v/v and analyzed. As shown in Figure 4, all 9 of the targeted components could be observed in the herbal supplement material.
Figure 4. Identification of Silymarin Components from a Commercial Natural Supplement
Stationary phase screening at the onset of method development, especially when dealing with a complex set of analytes, provides a facile means of analytical method development. As shown in the study, a few short experiments, coupled with powerful prediction software provided chromatographic conditions suitable for the analysis biologically active milk thistle compoenents. The developed conditions should prove useful in natural supplement, raw material and/or biological monitoring of silymarin complex components.

  1. Lee, J. I.; Hsu, B. H.; Wu, D.; Barrett, J. S. Journal of Chromatography A 2006, 1116, 57-68.

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