Spectrophotometric determination of L-α-glycerylphosphorylcholine in pharmaceutical formulations and industrial equipment cleaning rinse water with the WAKO Phospholipids C assay kit

Reagents and equipment

The WAKO Phospholipids C assay kit was purchased from Wako Diagnostics (Mountain View, CA, USA). It consists of the colour reagent containing phospholipase D, choline oxidase, peroxidase, 3,5-dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-sodium aniline and 4-aminoantipyrine and of the buffer solution, which composition and pH value are not specified. Native mold sn-glycerol-3-phosphocholine phosphodiesterase, in the form of the lyophilised powder containing tris(hydroxymethyl)aminomethane buffer salt, was purchased from Creative Enzymes (Shirley, NY, USA). The Hydranal moisture test kit for visual Karl-Fisher titration was purchased from Fluka (Buchs, Switzerland). Acetic anhydride (99%), methyl-4-hydroxybenzoate (99%) and propyl-4-hydroxybenzoate (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mercury (II) acetate (analytical grade), potassium hydrogen phthalate (analytical grade), perchloric acid (analytical grade), crystal violet (analytical grade) and glycerol (analytical grade) were purchased from Khimreaktivsnab (Tashkent, Uzbekistan). Glacial acetic acid was purchased from Lenreaktiv (Saint Petersburg, Russia). Choline alfoscerate (98%) was purchased from Lipoid GmbH (Ludwigshafen am Rhein, Germany). Different pharmaceutical formulations containing α-GPC were purchased from Sotex (Moscow, Russia). The flat plates made of stainless steel 12X12H10T were used to model the cleaning of industrial equipment. The analytical balance Sartorius Cubis MSA 225P-ICE-DI was used for weighting. The various micropipettes manufactured by Thermo Fisher Scientific (Waltham, MA, USA) were used for taking aliquots. The spectrophotometer Mettler Toledo UV7 was used for colorimetric measurements. The water bath Stegler WB-4 was used for sample incubations. The microburette Duran AS 5 ml was used for titration. The chemical glassware of the 2nd grade was used. Water for preparation of solutions was twice distillated and then deionised with Sartorius Arium Pro VF Ultrapure Water system.

Preparation of the colour reagent

The contents of one vial with the colour reagent and one vial with the buffer from the WAKO Phospholipids C assay kit, and the contents of one vial with sn-glycerol-3-phosphocholine phosphodiesterase were mixed together. The vials were rinsed with the resulting solution; the rinses were collected into the 50 ml volumetric flask, and the volume of the solution was adjusted by water. The solution was stored in the refrigerator.

Preparation of the 0.5% solution of crystal violet

A total of 0.50 g of crystal violet was weighted, dissolved in glacial acetic acid; the solution was transferred to the 100 ml volumetric flask, and the volume of the solution was adjusted by glacial acetic acid.

Preparation of the 3% solution of mercury (II) acetate

A total of 3.00 g of mercury (II) acetate was weighted, dissolved in glacial acetic acid, the solution was transferred to the 100 ml volumetric flask, and the volume of the solution was adjusted by glacial acetic acid.

Preparation and standardisation of the 0.1 M solution of perchloric acid

A total of 8.5 ml of perchloric acid was dissolved in ca. 900 ml of glacial acetic acid, then 30 ml of acetic anhydride was added, the solution was transferred to the 1,000 ml volumetric flask, and the volume of the solution was adjusted by glacial acetic acid. The water content in the solution was determined by visual Karl-Fisher titration using the Hydranal moisture test kit. If the water content was less than 0.1%, more water was added, and if it was greater than 0.2%, more acetic anhydride was added, and the water content determination was repeated. The solution was allowed to stand for 24 h. To standardise the solution 0.350 g of potassium hydrogen phthalate was weighted, dissolved in glacial acetic acid protected from light, and titrated with the perchloric acid solution using 0.05 ml of the 0.5% solution of crystal violet as indicator. This preparation procedure complies with State Pharmacopoeia of the Russian Federation (14th Edition) (2018).

Preparation of the 100 mg/l stock solution of α-GPC

A total of 1.0000 g of α-GPC was weighted, dissolved in glacial acetic acid, the solution was transferred to the 100 ml volumetric flask and the volume of the solution was adjusted by glacial acetic acid. The exact concentration of the solution was determined by titration. For this the aliquot of 5.0 ml of the prepared solution was transferred to the titration flask, 40 ml of acetic anhydride and 10 ml of the 3% solution of mercury (II) acetate was added, the solution was mixed and titrated with the standardised solution of 0.1 M perchloric acid using 0.05 ml of the 0.5% solution of crystal violet as indicator. Then the appropriate aliquot of the prepared solution with the determined concentration was taken, transferred to the 1,000 ml volumetric flask, and the volume of the solution was adjusted by water. The stock solution was stored in a refrigerator.

Preparation of working solutions of α-GPC

The working solutions of α-GPC with different concentrations ranging from 1 to 100 mg/l were prepared by appropriate dilution of the stock solution with water. The working solutions were prepared daily.

Preparation of sample solutions from injections

The solutions for intravenous injections available on the Russian local market contain a 250 g/l solution of α-GPC. The contents of ten ampoules from the single package were collected into a beaker; the aliquot of 5.0 ml was taken, transferred to the 500 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 5.0 ml of the prepared solution was taken, transferred to another 500 ml volumetric flask, and the volume of the solution was adjusted by water. The concentration of α-GPC in the resulting solution equals 25 mg/l.

Preparation of sample solutions from oral solutions

The oral solutions available on the Russian local market contain a 120 g/l solution of α-GPC. The contents of ten ampoules from the single package were collected into a beaker; the aliquot of 5.0 ml was taken, transferred to the 500 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 5.0 ml of the prepared solution was taken, transferred to another 500 ml volumetric flask, and the volume of the solution was adjusted by water. The concentration of α-GPC in the resulting solution equals 12 mg/l.

Preparation of sample solutions from capsules

The capsules available on the Russian local market contain 400 mg of α-GPC. The contents of ten capsules from the single package were collected into a beaker and dissolved in water; the solution was transferred to the 1,000 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 5.0 ml of the prepared solution was taken, transferred to the 500 ml volumetric flask, and the volume of the solution was adjusted by water. The concentration of α-GPC in the resulting solution equals 40 mg/l.

Preparation of model rinse water samples from injections

The contents of ten ampoules from the single package were collected into a beaker; the aliquot of 5.0 ml was taken, transferred to the 500 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 1.0 ml of the prepared solution was taken, placed onto the flat plate made of stainless steel 12X12H10T, and allowed to dry in the fume hood. The plate was rinsed several times with water, the combined rinses were transferred to the 100 ml volumetric flask, and the volume of the solution was adjusted by water. The expected concentration of α-GPC in the model rinse water sample equals 25 mg/l.

Preparation of model rinse water samples from oral solutions

The contents of ten ampoules from the single package were collected into a beaker; the aliquot of 5.0 ml was taken, transferred to the 500 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 1.0 ml of the prepared solution was taken, placed onto the flat plate made of stainless steel 12X12H10T, and allowed to dry in the fume hood. The plate was rinsed several times with water, the combined rinses were transferred to the 100 ml volumetric flask, and the volume of the solution was adjusted by water. The expected concentration of α-GPC in the model rinse water sample equals 12 mg/l.

Preparation of model rinse water samples from capsules

The contents of ten capsules from the single package were collected into a beaker; the aliquot of 5.0 ml was taken, transferred to the 500 ml volumetric flask, dissolved in water, and the volume of the solution was adjusted by water. The aliquot of 1.0 ml of the prepared solution was taken, placed onto the flat plate made of stainless steel 12X12H10T, and allowed to dry in the fume hood. The plate was rinsed several times with water, the combined rinses were transferred to the 100 ml volumetric flask, and the volume of the solution was adjusted by water. The expected concentration of α-GPC in the model rinse water sample equals 40 mg/l.

General procedure for the determination of α-GPC

A total of 3.0 ml of the colour reagent was mixed with 1.0 ml of working or sample solution of α-GPC in a test tube. The blank solution was prepared by mixing 3.0 ml of the colour reagent with 1.0 ml of water in another test tube. The contents of the test tubes were mixed, placed in the water bath and incubated at the temperature of 37 °C for 10 min. Then the absorbance of the working or sample solution of α-GPC at the wavelength of 595 nm in the glass cuvette with the optical path length one cm was measured against the blank solution.

Selection of the wavelength

The working solutions of α-GPC with the concentration of 30 mg/l and the blank solution were prepared and treated as described in the general procedure, and the spectrum of the working solution was recorded in the wavelength interval from 190 to 1,100 nm with the wavelength step of 0.2 nm in the glass cuvette with the optical path length of one cm against the blank solution. The spectrum is shown in Fig. 1. The maximum absorbance was observed at the wavelength of 595 nm. This wavelength was chosen for all further measurements.

Selection of the sample volume

The working solutions of α-GPC with the concentration of 30 mg/l and the blank solution were prepared as described in the general procedure. In the series of the test tubes, 3.0 ml of the colour reagent were mixed with the various volumes of the working solution ranging from 0.2 to 2.0 ml. The corresponding blank solutions were prepared in another series of test tubes by mixing 3.0 ml of the colour reagent with the various volumes of water. The solutions were incubated in the water bath at 37 °C for 10 min. The absorbances of prepared solutions with the different sample volume at the wavelength of 595 nm in the glass cuvette with the optical path length one cm were measured against the corresponding blank solutions. The results are shown in Table 2 and in Fig. 2. With the increase of the sample volume the molar concentration of α-GPC also increases, which favours the reaction rate to increase, but at the same point the molar concentrations of assay kit reagents and enzymes decrease by dilution, which favours the reaction rate to decrease. Therefore, at some point the reaction rate and the absorbance of the coloured product reach their maxima. The maximum absorbance was observed at the sample volume of 1 ml. This sample volume was chosen for all further measurements.

Selection of the incubation time

The working solutions of α-GPC with the concentration of 30 mg/l and the blank solution were prepared as described in the general procedure. In the series of the test tubes, 3.0 ml of the colour reagent were mixed with 1.0 ml of working solution. The blank solution was prepared by mixing 3.0 ml of the colour reagent with 1.0 ml of water in another test tube. The solutions were incubated in the water bath at 37 °C for the various time intervals ranging from 1 to 20 min. The absorbances of prepared solutions with the different incubation time at the wavelength of 595 nm in the glass cuvette with the optical path length one cm were measured against the blank solution. The results are shown in Table 3 and in Fig. 3. As one might see from Fig. 3, after 10 min the overall reaction becomes very slow, and the further absorbance increase becomes asymptotic, which makes it pointless to wait for longer. In contrast, taking a lesser period of time decreases the absorbance significantly and this reduces the sensitivity. The optimal absorbance was observed at the incubation time of 10 min. This incubation time was chosen for all further measurements.

Construction of the calibration curve

The working solutions of α-GPC with different concentrations ranging from 1 to 80 mg/l and the blank solution were prepared as described in the general procedure. In the series of the test tubes, 3.0 ml of the colour reagent were mixed with 1.0 ml of stock solution and prepared working solutions. The blank solution was prepared by mixing 3.0 ml of the colour reagent with 1.0 ml of water in another test tube. The solutions were incubated in the water bath at 37 °C for 10 min. The absorbances of prepared solutions with the different concentration of α-GPC at the wavelength of 595 nm in the glass cuvette with the optical path length one cm were measured against the blank solution. The results are shown in Table 4 and Fig. 4.

Analytical performance

The analytical performance of the method was determined in accordance with State Pharmacopoeia of the Russian Federation (14th Edition) (2018) guidelines. The method was tested for linearity, limits of detection and quantification, selectivity, accuracy, and inter- and intra-day precision.

Linearity

According to Fig. 2, the dependence of the absorbance of the coloured product at 595 nm on the concentration of α-GPC is linear in the range from 1 to 40 mg/l. The regression analysis was performed using the least-squares technique (Adrain, 1808). Additionally, the Ringbom’s optimum range (Ringbom, 1938; Ayres, 1949; Youmans & Brown, 1976), the molar attenuation coefficient, and the Sandell’s sensitivity coefficient (Sandell, 1944) were calculated. The parameters of the regression equation are listed in Table 5.

Limit of detection and limit of quantification

The limit of detection and the limit of quantification of the method were calculated according to Currie (1999), Shrivastava & Gupta (2011) and Little (2015). The values are presented in Table 5.

Selectivity with respect to common excipients

According to the Russian State Register of Pharmaceutical Products (https://grls.rosminzdrav.ru/Default.aspx), intravenous injections contain only a solution of α-GPC without excipients, oral solutions contain methyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, and capsules contain glycerol as the common excipients. The possible interference of these excipients as well as of acetic acid (because it was used in the stock solution preparation) was studied. For that, the 50 mg/l solutions of methyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, and the 1% solutions of glycerol and acetic acid were prepared. 3.0 ml of each solution were placed in the test tubes, 1.0 ml of the colour reagent was added to each one, and the solutions were incubated in the water bath at the temperature 37 °C for 60 min. No colour development was observed; this indicates that the tested excipients did not interfere.

However, the implementation of the WAKO Phospholipids C assay kit for the analysis of α-GPC in complex matrices is not possible. Blood and other bodily fluids contain phospholipids, which are also affected by this kit, and in this case it quantifies the phospholipids instead of α-GPC. Because one of the reaction steps includes the formation of hydrogen peroxide, any other enzymes and substrates that produce H₂O₂ also interfere. This implies that this method is not suitable for the determination of α-GPC in food and plant material, because many of raw natural ingredients contain both glucose and glucose oxidase, which lead to the H₂O₂ production.

Accuracy

Four series of experiments were conducted. In the first series ten working solutions with the concentration of α-GPC equal to 5 mg/l, in the second series ten sample solutions from injections with the concentration of α-GPC equal to 25 mg/l, in the third series ten sample solutions from oral solution with the concentration of α-GPC equal to 12 mg/l, and in the fourth series ten sample solutions from capsules with the concentration of α-GPC equal to 40 mg/l were prepared. The solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded; the concentrations of the solutions were calculated according to the regression equation, and the relative uncertainties were determined. The results are collected in Table 6.

Intra-day precision

Four series of experiments were conducted. In the first series ten working solutions with the concentration of α-GPC equal to 5 mg/l, in the second series ten sample solutions from injections with the concentration of α-GPC equal to 25 mg/l, in the third series ten sample solutions from oral solution with the concentration of α-GPC equal to 12 mg/l, and in the fourth series ten sample solutions from capsules with the concentration of α-GPC equal to 40 mg/l were prepared. The solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded; the concentrations of the solutions were calculated according to the regression equation, and the relative standard deviations were determined. The results are collected in Table 7.

Inter-day precision

Four series of experiments were conducted. In the first series a working solution with the concentration of α-GPC equal to 5 mg/l, in the second series a sample solution from injections with the concentration of α-GPC equal to 25 mg/l, in the third series a sample solution from oral solution with the concentration of α-GPC equal to 12 mg/l, and in the fourth series a sample solution from capsules with the concentration of α-GPC equal to 40 mg/l were prepared each day during consecutive five days. The solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded; the concentrations of the solutions were calculated according to the regression equation, and the relative standard deviations were determined. The results are collected in Table 8.

Accuracy for the determination of model rinse water samples

Three series of experiments were conducted. In the first series five model rinse water samples from injections with the concentration of α-GPC equal to 25 mg/l, in the second series five model rinse water solutions from oral solution with the concentration of α-GPC equal to 12 mg/l, and in the third series five model rinse water solutions from capsules with the concentration of α-GPC equal to 40 mg/l were prepared. The solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded; the concentrations of the solutions were calculated according to the regression equation, and the relative uncertainties were determined. The results are collected in Table 9.

Precision for the determination of model rinse water samples

Three series of experiments were conducted. In the first series five model rinse water samples from injections with the concentration of α-GPC equal to 25 mg/l, in the second series five model rinse water samples from oral solution with the concentration of α-GPC equal to 12 mg/l, and in the third series five model rinse water sample solutions from capsules with the concentration of α-GPC equal to 40 mg/l were prepared. The solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded; the concentrations of the solutions were calculated according to the regression equation, and the relative standard deviations were determined. The results are collected in Table 10.

Comparison of the results of titrimetric and spectrophotometric determinations

A total of 1.0000 g of α-GPC was weighted, dissolved in ca. 80 ml of glacial acetic acid, the solution was transferred to the 100 ml volumetric flask and the volume of the solution was adjusted by glacial acetic acid. Ten aliquots of 5.0 ml of the prepared solution were transferred to the titration flasks, 40 ml of acetic anhydride and 10 ml of the 3% solution of mercury (II) acetate was added to the each, the solutions were mixed and titrated with the standardised solution of 0.1 M perchloric acid using 0.05 ml of the 0.5% solution of crystal violet as indicator. Then ten aliquots of 5.0 ml of the prepared solution were taken, transferred to ten 2,000 ml volumetric flasks, and the volumes of the solutions were adjusted by water. The diluted solutions were treated as described in the general procedure, and then the absorbance of the coloured product was recorded, and the concentrations were calculated according to the regression equation. The content of α-GPC in the weighting was calculated using the both methods, and the F-test of equality of variances and the t-test of equality of means were performed. The results are shown in Table 11. As could be seen, both the calculated F- and t-values do not exceed the critical values for the given degrees of freedom and p-value, which means that these titrimetric and spectrophotometric methods give statistically equal results for the tested sample.