Development of thin-layer chromatography‒densitometry for the quantification of lecithin in dietary supplements

2.1 Instrumentation

Linomat 5 sample applicator, densitometer TLC Scanner 3 with winCATS4 software, and UV lamp (254/366 nm) were manufactured by CAMAG (Muttenz, Switzerland). Laboratory drying oven Ecocell was produced by BMT Medical Technology (Brno, Czech Republic), and analytical balance WPA 120/C/1 by Radwag (Radom, Poland). HPTLC silica gel 60F254, TLC silica gel 60F254 and HPTLC cellulose chromatographic plates were purchased from Merck (Darmstadt, Germany).

2.2 Reagents and standard substance

Ammonia, butylamine, ammonium molybdate tetrahydrate (No. A7302), ninhydrin (No. 151173), iodine (No. 207772) and L-α-phosphatyidylcholine (No. P3556) were purchased from Sigma-Aldrich (Poznań, Poland). Chloroform, ethanol, glacial acetic acid, sulfuric acid 95%, and methanol were purchased from Chempur (Piekary Śląskie, Poland).

2.3 Examined preparations

Thirteen products containing lecithin were tested. Two of them were registered as drugs, and eleven are a dietary supplements. All preparations and supplements were purchased at local pharmacies. Some of the preparations have exceeded the expiry date within the time of the study. The following preparations were analyzed: Products A‒F, H, and K‒N, swallow capsules containing 1200 mg of lecithin; Product G, chewable dragees containing 750 mg of lecithin; and Product I, liquid containing 10.4 g of lecithin per 100 mL.

2.4 Standard and sample solutions

Standard solution of L-α-phosphatidylcholine with a concentration of 0.1% (m/V) was prepared by dissolving the substance in methanol.

The contents of five capsules/dragees were weighed, and the average weight of a single capsule/dragee was calculated. The appropriate amount (to obtain solutions with a concentration of approx. 0.1%) of each preparation was weighed or the appropriate volume of the liquid was taken, assuming the lecithin content declared by the manufacturer. Each of the samples was dissolved in methanol in 25 mL volumetric flasks, that were left in the refrigerator overnight, to fully extract the PC from the preparation mass. Next, the solutions were filtered through cellulose filters; the obtained filtrate was used for further studies.

2.5 Staining reagent

Ammonium molybdate solution (0.5%, m/V) was prepared in a mixture of ethanol‒25% aqueous) H2SO4 in a 1:9 volume ratio.

2.6 Optimization of the analysis conditions

In order to select the appropriate mobile phase, solvent mixtures of varying proportions were tested. In order to determine the optimal composition, after performing preliminary tests and analyzing the available literature, it was decided to use a mixture of chloroform and methanol with the addition of various amounts of ammonia or acetic acid. Each of the mobile phases was tested by developing plates with two bands from the standard solution and sample solution (PC concentration about 0.1%). In addition, different stationary phases (TLC silica gel 60F254, HPTLC silica gel 60F254, HPTLC cellulose) were tested. Detection of PC in the UV range turned out to be impossible, therefore it was necessary to carry out a color reaction with the components on the chromatographic plates. Several detection methods were tested, such as staining in iodine vapor (aqueous solution of iodine 0.05 mol/L), spraying with sulfuric acid, spraying with 5% (m/V) solution of ammonium molybdate in 25% aqueous sulfuric acid, spraying with 5% (m/V) solution of ammonium molybdate in ethanol with the addition of sulfuric acid, immersion in 5% (m/V) ethanolic solution of ammonium molybdate with the addition of sulfuric acid, spraying with 0.2% (m/V) solution of ammonium molybdate in ethanol with the addition of sulfuric acid, spraying with 0.5% (m/V) ammonium molybdate in ethanol with sulfuric acid, or spraying with 0.2% (m/V) ninhydrin solution [60,61,62]. The effects of some of the performed experiments are presented in Table S2.

Based on performed tests a mixture of chloroform‒methanol‒glacial acetic acid (3:6:0.4, V/V) was selected as the mobile phase. On cellulose plates after applying the solutions, developing and visualization, two blurred streaks were obtained, which made it impossible to identify. The use of TLC and HPTLC plates allowed to obtain the chromatograms with no differences in the position of the spots, and the time of analysis. Consequently, the cheaper TLC 60F254 plates were chosen. And finally, for visualization of the chromatograms, a 0.5% (m/V) ammonium molybdate solution in a mixture of ethanol and H2SO4 (1:9, V/V) was selected for staining by spraying on the plates, and then drying at 100 °C for 5 min. The reagent was prepared immediately before use. In effect, colored gray-blue bands were obtained on a white background. The plates were then densitometrically scanned at the selected wavelength.

2.7 Sample preparation

The effect of PC extraction time on its efficiency was investigated. For this purpose, one of the preparations (Product L) was weighed (approx. 0.02 g) into 25 mL flasks, dissolved in methanol, and shaken for 5, 15, and 30 min, respectively. The solutions were filtered, and applied to the TLC 60F254 plates with the Linomat 5 applicator in various volumes. A PC standard solution in a volume of 5 µL was also applied. The plates were placed into a chromatographic chamber saturated with a chosen earlier mobile phase. After development, the chromatogram was drying and sprayed with a staining reagent. The plates were dried at 100 °C for 5 min, and then scanned densitometrically. Moreover, an analogous sample was prepared, which was left in the refrigerator overnight after being filled with methanol. After applying the obtained solution and developing the chromatogram, it was found that the registered peak areas were larger. Thus, it was found that the extraction time had a great influence on its effectiveness. Therefore, the following extraction time was selected for further analysis: 15 min of shaking with methanol, 24 h in the refrigerator, 5 min of shaking, and filtering.

2.8 Conditions of the TLC–densitometry assay

In order to analyze the PC content in pharmaceutical products and dietary supplements, methanol solutions of preparations were prepared with a concentration of about 0.1% (m/V) in relation to the amount of lecithin declared by the manufacturer. For this purpose, the appropriate mass or volume (in the case of liquids) of the preparations were weighed into 25 mL flasks. Methanolic suspensions were prepared as described above (Sect. 2.7) and then filtered through cellulose filters. For each preparation, three series of samples were made, prepared in the same way from three independent weights. The solutions were applied with a Linomat 5 applicator at a rate of 650 nL s−1 to TLC 60F254 silica gel chromatography plates, 15 × 10 cm in size. On each plate the preparation and standard solutions (in a volume of 15 and 5 µL) were placed. The bands were 0.7 cm wide, and were placed 1 cm from the bottom and left edges of the plate. Next, chromatograms were developed in the chamber saturated for approx. 15 min with the mobile phase containing chloroform‒methanol‒glacial acetic acid (3:6:0.4, V/V), on a 9 cm path, in approx. 30 min. After drying, the chromatogram was sprayed with 0.5% (m/V) ammonium molybdate in mixture of 25% aqueous sulfuric acid and ethanol (1:9, V/V), prepared immediately before use. The plates were dried for 5 min at 100 °C, and then scanned with a TLC Scanner 3 densitometer in absorption–reflection mode at λ = 360 nm. The densitometric scanning (winCATS 1.4.8v. software) conditions were as follows: slit dimensions 5.00 × 0.45 mm, scanning speed 20 mm s−1, data resolution 100 μm per step. Based on the recorded densitograms, the retardation factor (RF) for PC was designated to be 0.23. The values of the obtained peak areas for the sample and standard solutions were used for quantitative analysis.

2.9 Validation of the method

Method validation is an essential element in assessing the suitability of an analytical procedure to generate reliable results [63]. The validation process is carried out by checking the parameters of the method using a number of appropriate indicators, i.e., accuracy, precision, linearity, range, limit of detection (LOD), and limit of quantification (LOQ).

2.9.1 Linearity

The linearity of an analytical method is the ability to obtain results that are proportional to the content (or concentration) of the analyte in the sample. In order to determine the linearity of the developed method, standard substance solutions were prepared at the concentrations in the range from 0.23 to 3.21 mg mL−1. Each of the solutions was applied to the TLC plate in the same volume. Based on the obtained results, the relationship P = f(c) was drawn, and the parameters of linear regression were determined. Then, residual analysis was performed, which allows to determine if the selected model was properly fitted.

2.9.2 Sensitivity

The limit of detection (LOD; the smallest amount of an analyte in a sample that can be detected) and the limit of quantification (LOQ; the smallest amount of an analyte in a sample that can be quantified with sufficient precision and accuracy) were determined according to the equations:

and

where: Sb = standard deviation of the intercept, a = the slope of the calibration curve.

2.9.3 Precision

The precision of an analytical method (repeatability, reproducibility, intermediate precision) is the degree of dispersion of the results obtained in a series of measurements on the same homogeneous sample, using the same method, under the described conditions. The evaluated parameters for precision are: standard deviation or relative standard deviation (RSD, %).

2.9.4 Accuracy

The accuracy of the analytical method determines the degree of agreement between the value considered to be true, and the content of the substance in the test sample obtained during the analysis. It should be determined at 3 concentration levels, based on a minimum of 9 test results. The calculation for accuracy (R, as % recovery) was calculated according to the formula:

$$ R\left[ \% \right] = 100 \cdot \frac } \right) - \left( x \right)}}, $$

where: x = content in the sample without addition of standard, x + si = content in the sample with addition of standard, s = added amount of standard.

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