Phospholipid quantification

Several methods for quantification of phospholipids were investigated using HPLC, TLC and the Stewart colourimetric assay. HPLC analysis could only be conducted on non-hydrogenated phospholipids as the lack of double bonds in the hydrogenated forms prevents the detection by conventional UV absorbance. HPLC can be conducted on hydrogenated phospholipids through the use of an ELDS detector which was not feasible for this project.

Quantitative TLC analysis was investigated as a means of quantification of phospholipids. The mobile phase consisted of chloroform:methanol:water (65:25:4) (Torchilin and Weissig, 2003). Standards of hydrogenated soy phosphatidylcholine (HSPC) were prepared in chloroform to final concentrations of 10, 5, 2.5, 1.25 and 0.625 mg/ml by serial dilution of a stock standard that was prepared by dissolving 50 mg of HSPC in 5 ml chloroform. Silica gel TLC plates (TLC Silica gel 60 F254, Merck,

Darmstadt, Germany) were prepared by cutting into it 7 × 10 cm plates and drawing a horizontal pencil line, 1 cm from the base of the plate. 10 l of the standard solutions were spotted onto the line, 1.5 cm apart and allowed to air dry for 1 hour prior to analysis. Plates were developed in chromatography tanks, equilibrated with the mobile phase until the solvent front reached 1 cm from the top, removed from the tanks and air dried prior to staining. Plates were stained by a 1 sec immersion into rhodamine B stain solution and allowed to dry overnight. Scanned TLC plates were then analysed using Sorbfil Version 1.0 TLC visualiser software. Figure 3.25 below shows an example of the chromatogram derived from the TLC plate.

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Figure 3.25: Chromatogram derived from the TLC plate using Sorbfil version 1.0

densitometry software.

The standard curve for phospholipids using this method is shown in Figure 3.26 and displayed a near linear relationship with a linear correlation coefficient (r2) of 0.9970. The y-intercept for the linear graph was 6.59% of the 50% response, which is higher than the recommended 2%.

0 5 10 15 0 20000 40000 60000 80000 100000 Phospholipid Concentration (mg/ml) P e a k A re a

Figure 3.26: Standard curve for quantitative TLC determination of hydrogenated soy

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Apart from the difficulty in obtaining reliable standard curve data, it was also noted by observation that the tail group of the phospholipid influences the size of the spot on the chromatogram and the use of various chain lengths (as would be necessary during the optimization phase) would be unlikely to produce accurate results. The assay for phospholipid required that quantification of the phospholipid could be performed that was only dependent on the head group and not the rest of the molecule.

Of the colourimetric assays that rely on interaction with organic phosphates, the most extensively reported assays are the Stewart assay (Stewart, 1980) and the Bartlett assay (Torchilin and Weissig, 2003). Of these two, the most favoured is the Stewart assay, as it only quantifies organic phosphates and not inorganic phosphates such as those found in phosphate buffered saline (PBS), which was extensively used as a hydration buffer during liposome manufacture. The Stewart assay uses a dye, ammonium ferrothiocyanate, which is insoluble in chloroform, but when complexed with the phosphate portion of a phospholipid, becomes soluble in chloroform and partitions from the aqueous phase into the organic chloroform phase. The absorbance of the complex dye, with a λmax of 488 nm, is easily measured using a

standard UV/VIS spectrometer and is far more rapid than those methods that measure inorganic phosphates after a lengthy digestion step (Stewart, 1980).

The Stewart assay was therefore conducted as described previously (Torchilin and Weissig, 2003). A solution of ammonium ferrothiocyanate was prepared through the reaction of 27.0 g of ferric chloride with 30.4 g of ammonium thiocyanate in 1000 ml RO water and the solution freshly prepared on a weekly basis. In order to accurately quantify the phospholipids, a calibration curve was prepared using standard solutions of phosphatidylcholine in chloroform. A stock solution containing 0.24 mg/ml of phosphatidylcholine was prepared in chloroform and diluted according to the scheme shown in Table 3.5. The assay was performed by the addition of 2.0 ml of

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chloroform, containing a proportion of phospholipid standard, to 2.0 ml of ammonium ferrothiocyanate solution in a 10 ml glass test tube. The mixture was vortexed for 15 seconds and centrifuged at 1000 rpm for 5 minutes to separate the aqueous phase from the organic phase. The optical density of the lower, organic, layer, was read on a spectrophotometer at 485 nm. Standards were triplicated and a standard curve constructed. Linear regression analysis was performed, yielding a linear correlation coefficient (r2) of 0.9990, which was considered to be linear (Figure 3.27). Analysis of the y-intercept to determine if the intercept was not significantly different from the origin, showed that the y-intercept was 0.69% of the 50% response, which is well below the recommended 2% (Ahuja and Dong, 2005).

Table 3.5: Standard preparation for the determination of phospholipid concentration.

Tube Standard Volume * Chloroform Ammonium Ferrothiocyanate Phospholipid Concentration 1 0.0 ml 2.0 ml 2.0 ml 0 mg/ml 2 0.1 ml 1.9 ml 2.0 ml 0.0075 mg/ml 3 0.2 ml 1.8 ml 2.0 ml 0.015 mg/ml 4 0.4 ml 1.6 ml 2.0 ml 0.03 mg/ml 5 0.6 ml 1.4 ml 2.0 ml 0.06 mg/ml 6 0.8 ml 1.2 ml 2.0 ml 0.09 mg/ml 7 1.0 ml 1.0 ml 2.0 ml 0.12 mg/ml

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0.00

0.05

0.10

0.15

0.0

0.5

1.0

1.5

Concentration (mg/ml)

A

b

s

o

rb

a

n

c

e

@

4

8

5

n

m

Figure 3.27: Standard curve for colorimetric determination of phospholipids in liposome

suspensions (n = 3 for each point). r2 = 0.9990, y = 10.71x - 0.00451.

Liposome suspensions were analysed for phospholipid content as follows. 50 l of liposome suspension was added to 2 ml of chloroform and vortexed for 30 seconds in order to extract the phospholipid from the aqueous suspension. The assay was then conducted as discussed above, with the addition of 2 ml ammonium ferrothiocyanate, vortex mixing for a further 15 seconds and measurement of the absorbance of the lower organic phase at 485 nm. Concentration in the suspension was determined from the standard curve and multiplying the concentration of phospholipid in the chloroform by the dilution factor (40).