Determination of free phthalic acid by HPLC

Figure 2.17: UV-spectrum of phthalic acid (5.96 mg/100 ml) in a mixture of MeOH and water (50+50 ml).

Figure 2.18: UV-spectrum of a standard mixture of phthalic acid (4.864 mg/100 ml) and nicotinamide (0.752 mg/100 ml) in a mixture of MeOH and water (50+50 ml).

0.0 0.2 0.4 0.6 0.8 1.0 1.2

250 260 270 280 290 300 310 320

Wavelength (nm)

Absorbance

0.0 0.5 1.0 1.5 2.0 2.5 3.0

209 229 249 269 289 309

Wavelength (nm)

Absorbance

Some authors <155,20> have reported about the determination of free phthalic acid by using HPLC-method. Therefore in this work the modified method based on the former reports was used. The 0.025 M phosphate buffer <125> in mixture with MeOH in different ratio was used as eluent. Different flow rates were applied as trials in order to reduce the retention time of the substances. However, the conditions should not be performed with a high pressure to maintain the life of the column and other parts of the

system. Three samples, i.e. phthalic acid (1.12 mg/100ml), nicotinamide (1.06 mg/100ml) and their mixture were prepared. If nescessary the pH {53} was

adjusted below 3 using ortho-phosphoric acid {Ortho-phosphoric acid 85 0%, p.a. grade, Lot No. 83560, Riedel-de-Haen, Seelze}. Each solution was filled in HPLC vials with caps {Vials N11-1, Art No. 70214, Amber glass 1 ml, Macherey-Nagel, Düren} then measured by different conditions demonstrated in Table 2.7. The best suitable condition was then selected basing on the presented low pressure and the well separated peaks of nicotinamide and phthalic acid with an acceptable retention time. The example of the HPLC-chromatogram was shown in Figure 2.19. At the region of 3.0 min ( O ) were peaks of the injection, at 4.4 min ( I ) was a peak of nicotinamide and at 10.8 min ( II ) was a peak of phthalic acid.

Figure 2.19: HPLC chromatogram of a standard mixture containing nicotinamide 2.15 mg/100ml ( I ) and phthalic acid 1.92 mg/100ml ( II ) at a flow rate of 0.8 ml/min and a mixture of 0.025 M phosphate buffer and MeOH (80+20 ml) as an eluent.

O

I

II

Substance Mobile phase Flow rate Pressure Retention time

(ml+ml) (ml/min) (bar) (min)

buffer * + MeOH nicotinamide phthalic acid

nicotinamide 80 + 20 1.2 270 2.9 -

1.0 230 3.5 -

0.8 187 4.4 -

phthalic acid 80 + 20 1.2 270 - 7.1

1.0 230 - 8.6

0.8 187 - 10.8

std mixture 80 + 20 1.2 270 2.9 7.1

1.0 230 3.5 8.6

0.8 187 4.4 10.8

nicotinamide 70 + 30 0.8 - 3.6 -

phthalic acid 0.8 - - 7.2

std mixture 0.8 - 3.6 7.2

nicotinamide 50 + 50 0.8 - 3.2 -

phthalic acid 0.8 - - 4.1

std mixture 0.8 - 3.2 4.1

Table 2.7: Conditions of HPLC in the preliminary trials; nicotinamide

(1.06 mg/100 ml), phthalic acid (1.12 mg/100ml), std mixture contained nicotinamide (2.15 mg/100 ml) and phthalic acid (1.92 mg/100ml), buffer * = 0.025 M phosphate buffer <125>, and - means not measured

The following conditions will therefore be used for characterization of free phthalic acid in coated pellets containing nicotinamide.

HPLC conditions:

Column {28}: Nucleosil 5µ C18, 100 °A Dimensions: 250 x 4.6 mm

Mobile Phase: 0.025 M Phosphate Buffer-Methanol (80 + 20 ml, pH 3) Flow rate {31}: 0.8 ml/min

Detection {30}: UV @ 210 nm Temperature: ambient Injection {27}: 20 µL

2.2.15.1 Calibration and validation

Calibration

The conditions as mentioned above were applied to create a calibration curve of phthalic acid in water. The mobile phase containing 0.025 M phosphate buffer and methanol was first filtered through the filter set containing a cellulose nitrate filter paper {Lot No. 0899113079901503, 0.2 µm, Sartorius, Göttingen} and then the solution was degassed by putting the volumetric flask into the ultrasound bath {67} for 15 min. This solution of mobile phase was pumped {31} through the degas unit {29} into the HPLC-column {28}.

The solution of the standard substance {Phthalic acid, C8H6O4, Lot No. 42130, Riedel-de-Haen, Seelze} was prepared by dissolving free phthalic acid in CO2-free water and the pH was adjusted below 3 using ortho-phosphoric acid {Ortho-phosphoric acid 85 %, p.a. grade, Lot No. 83560, Riedel-de-Haen, Seelze}. The solutions of 8 different concentrations of 0.34, 0.51, 0.68, 1.02, 1.53, 5.00, 10.00, 17.00 mg/100 ml were prepared in triplicate and each concentration was measured three times. At the end there were 72 values for calculation of the regression line. After the test of linearity and homogenity at the confidential level of 95 % using the statistical programm Toccata {S4}, the regression line using the area under the curve of HPLC-chromatogram showed a slope of 1114782 with a standard deviation of ± 2153. The limits of detection and determination were 0.02309 mg/100ml and 0.14064 mg/100ml, respectively. On the other hand the regression line using the height of the curve of HPLC-chromatogram showed a slope of 69.77 with a standard deviation of ± 0.144. The limits of detection and determination were 0.02899 mg/100ml and 0.15723 mg/100ml, respectively. The example of the residual plot using the area of 72 values was shown in Figure 2.20.

Validation

The validation was performed by measuring the known standard solution of phthalic acid (2.0 mg/100ml) and nicotinamide (2.0 mg/100ml) in CO2-free water for three days.

Each solution was freshly prepared at every day of the measurement in triplicate and every solution was measured three times. The measured values of the area under the curve or the height were calculated to receive the concentration. The data calculated by using regression line based on the area under the curve gave the values of recovery of 99.0 ± 0.1 %, whereas the values using the regression line based on the height of the

peak gave the values of recovery of 98.0 ± 0.5 %. This means this method is suitable for detecting of free phthalic acid in a mixture with nicotinamide.

Figure 2.20: Residual plot of 72 values of the calibration curve of the solution of phthalic acid in water, using HPLC-method.

2.2.15.2 Measurement of free phthalic acid in coated pellets

The exact amount of about 2.50 g of enteric coated pellets (Product Ff, Table 2.27) was weighed {2} and then they were put in a 100 ml-volumetric flask, 10 ml of tetrahydrofuran {Tetrahydrofuran, LiChrosolv, Lot No. I 912101949, Merck KGaA, Darmstadt} was added, followed by about 70 ml of distilled water. The samples were vibrated in the ultrasound bath for 30 min. The pH of the dispersion was adjusted to lower than 3 by adding ortho-phosphoric acid {Ortho-phosphoric acid 85%, p.a. grade, Lot No. 83560, Riedel-de-Haen, Seelze} and then the total volume of 100 ml was adjusted by distilled water. After well mixing the dispersion was filtered through 0.45 µm PTFE-filter {Rotilabo Spritzenfilter, Art No. P8161, 0.45 µm, Carl Roth, Karlsruhe}

before analysis by HPLC at the condition as mentioned before. Five samples were prepared after the above mentioned method. Every sample was measured three times and therefore at the end there were 9 values for one product. The mean value of free

-100000 -50000 0 50000 100000 150000 200000 250000

0 5 10 15 20

Concentration of phthalic acid in water (mg/100ml)

Residual of area

phthalic acid in the samples was calculated using both the regression lines based on the area and the height of the HPLC-chromatograms.