Thin Layer Chromatography

Bile Acid Separation

In contrast to other analytical technologies such as HPLC and gas chromatography, which are also com monly used for bile acid analysis, TLC is a very simple and nearly universal method. No highly sophisticated and expensive instrumentation is needed for reliable semiquantitative analysis. If precise quantification is essential, a TLC scanner equipped with a computer software system for fast and easy data registration and evaluation is recommended. Whereas HPLC analysis is limited by the fact that only one sample can be analysed at a time, TLC allows multiparallel analysis of probes in combination with an almost unlimited number of detection and visualization methods.

Several solvent systems for the separation of bile acids in biological fluids are described. With respect to bile, bile acid concentrations are in the millimolar range and therefore no detection problems occur, which makes TLC well suited for bile and bile acid analysis. In addition, time-consuming sample pre-treatment procedures can be generally avoided for standard analysis of bile acids. However, dilution of the bile samples with phosphate-buffered saline increases the sensitivity of the selected detection (Figure 4).

For bile acid pattern analysis a satisfactory separation of the main components, the glycine- and taurine-conjugated bile acids, is necessary. In general, silica gel TLC plates are used for routine analysis since high quality materials are commercially available in several sizes and from several suppliers and, in contrast to reversed-phase plates for example, at a relatively low price. Our experience in this field leads us to suggest the use of silica gel pre-coated on glass plates with a concentration zone to improve separation, bile acid band shape and overall resolution. Solvent systems suitable for the separation of bile acids on silica gel are shown in Table 1. Many methods for bile acid separation have been described in the literature, and only a selection are shown here.

System 2 leads to the most satisfactory results in resolution and band shape. With this system the glycine and taurine conjugates of deoxycholic acid and of chenodeoxycholic acid can be separated with good resolution. The TLC plates have to be developed six times in the same solvent mixture for bile acid separation and this requires more time compared with the other solvent systems described, but if quantification by TLC scanning is followed system 2 is the method of choice. Solvent system 3 provides a simple and fast method for the separation of conjugated and unconjugated bile acids with sufficient overall resolution to allow quantification. The advantage of this system is the simultaneous separation of cholesterol, which migrates faster than the bile acids. Chamber saturation before analysis increases resolution and improves band shape. With system 4, a solvent

Potassium 1.2% Calcium 0,4%

Chloride 33.1%

Bile acids 70.1%

Figure 3 Bile acid patterns obtained from three samples of human T-drainage bile and from the bile of other animals. Key as for Figure 1, plus GDC, glycodeoxycholic acid and TDC, taurodeoxycholic acid. (From Muller etal. 1992.)

mixture for the separation of conjugated bile acids on reversed-phase TLC plates is provided. TLC conditions are similar to an HPLC method described for the separation of conjugated bile acids, and may be helpful in comparison of sample analysis by different analytical techniques or in cases where simultaneous separation of bile acids and drug metabolites is required.

In all the separation systems described temperature plays an important role in resolution.

Although room temperature (20-22°C) allows satisfactory separation employing the systems described, in some cases decreasing the temperature improves separation and band shape. Depending on the sample concentration and composition the influence of temperature on separation resolution should be taken

20 40 60 80 100 120 140

Figure 4 (A) Thin-layer chromatogram of increasing amounts (1, 2 and 4 ^L of 4 mg mL~1 solutions) of bile acid standards TC, GC, TLC, GCDC, C, GLC, UDC and LC. (B) Fluorescence scan of the left lane of the chromatogram. Key as for Figure 1. (From Muller etal. 1992.)

20 40 60 80 100 120 140

Figure 4 (A) Thin-layer chromatogram of increasing amounts (1, 2 and 4 ^L of 4 mg mL~1 solutions) of bile acid standards TC, GC, TLC, GCDC, C, GLC, UDC and LC. (B) Fluorescence scan of the left lane of the chromatogram. Key as for Figure 1. (From Muller etal. 1992.)

into account as a critical factor for optimization of the chosen solvent system.

Numerous detection methods for bile acid visualization on TLC plates after separation have been described and two principally different systems are in use. Detection methods using reagents which form coloured complexes have the advantage that bands can be visualized directly. A universal detection re agent for a vast amount of biological substances is anise aldehyde dissolved in sulfuric acid. Spraying or dipping of the developed TLC plates followed by incubation at 125°C generates spots of different colour which are stable for some time. However, the low specificity of this reagent may cause problems in the analysis of samples with a high content of other components or analytes. Spots can also be visualized under ultraviolet (UV) light, which increases sensitivity. Individual bile acids can be identified by their different colours (Figure 5).

Molybdatophosphoric acid is another reagent often used for detection of biological components and forms blue bands on a yellow ground. Since colour intensity of the visualized spots is not stable over a long period of time, quantification must follow immediately after development of the colour.

The sensitivity of detection of bile acids on TLC plates can be increased dramatically by using a reagent system which forms fluorescent bands. HClO4 has been described as giving reproducible fluorescent spots with bile acids on TLC plates, provided a 5% solution of this derivatizing agent is used and UV light of 365 nm excitation wavelength is employed after spraying and appropriate treatment at higher temperature.

According to our experience a reagent mixture of manganese dichloride and sulfuric acid is preferred for detection, owing to the reproducible results and its convenience in use. Plates are dipped in the reagent

Table 1 Solvent systems for bile acid separation

System 1

Unconjugated bile acids

System 2

Glycine- and taurine-conjugated bile acids

System 3 All bile acids

System 4

Glycine- and taurine-conjugated bile acids

Isooctane Diethyl ether n-Butanol Acetic acid

Chloroform 2-Propanol 2-Butanol Acetic acid Double-distilled water

Chloroform Methanol Acetic acid

Acetonitrile (90%)

0.01 M Ammonium carbamate pH 7.3

Room temperature

Room temperature 2x15 cm followed by 4x7 cm

Room temperature Chamber saturation

Room temperature Separation on RP8 plates

Figure 5 Fluorescence scans of bile specimens from Figure 1: (A) human bile II; (B) human bile I; (C) rabbit bile; (D) pike bile; (E) monkey bile; (F) carp bile; (G) ox bile. Key as for Figure 1. (From Muller etal. 1992.)

and heated to 110°C for 10-15 min. Bile acids react with the reagent with the formation of yellow or brown coloured spots. Under UV light (365 nm) blue or yellow fluorescing bands can be detected. Bile acids can be differentiated by their respective colour. The detection sensitivity for the bile acids under UV con ditions is 2-5 ng and is therefore about 1000-fold higher compared to visible light (Figure 4).

Cholesterol

Cholesterol and cholesterol esters are important bile components. Several solvent systems for the

Table 2 Solvent systems for separation of cholesterol and cholesterol esters on silica gel plates

System 1 Chloroform Methanol Acetic acid

System 2

Chloroform

Acetone

System 3 Cyclohexane Diethyl ether

System 4 Methanol Diethyl ether n-Hexane

Room temperature Chamber saturation

Room temperature

Room temperature

Multiple plate development separation of these steroids exist, but only some of them are suitable for the analysis of cholesterol in complex biological fluids. In Table 2 four commonly used methods are shown. The mixture of chloroform and acetone is described for the detection of cholesterol in serum samples and can successfully be adapted to bile analysis. In system 4 three organic solvents with decreasing polarity are suggested for three consecutive development steps and can be used in automated multiple development devices.

If simple and fast one-step analysis of cholesterol and bile acids is required, solvent system 1 combined with the above described manganese detection reagent should be used, since cholesterol can be separated and identified easily by fast migration and its orange fluorescence. Other staining reagents containing either trichloracetic acid or 8-anilinonaphthaline-sulfonic acid ammonium salt have been shown to react with cholesterol to fluorescent bands, and numerous other universal detection systems are described in the literature. Adaptation to bile analysis seems to be possible in most cases.

Use of the NCS reagent (1,2-naphthochinone-2-sulfonic acid sodium salt) leads to purple bands which can be easily detected and quantified with detection limit at 5 ng cholesterol.

Phospholipids

As for the other bile components, several solvent systems for separation of phospholipids are known. However, most of them were not developed for the analysis of bile samples, but for other biological ma terials or mixtures of purified standard compounds. Due to the fact that the most abundant phospholipid in bile samples is lecithin, three solvent systems able to separate phospatidylcholine, phosphatidyl-ethanolamine, phosphatidylserine and phosphatidyl-glycerol are shown in Table 3.

Some of the most commonly used and versatile detection reagents described in the sections above are also suitable for phospholipid detection both under UV and visible light. Detection with the 2,7-difluores-cein reagent increases sensitivity and allows reliable and satisfactory quantification. Detection by a reagent mixture containing ammonium molybdate, sul-furic acid and ascorbic acid results in blue bands which can be easily detected and quantified by a TLC scanning device at a wavelength of 620 nm.

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