Spectrofluorimetric determination of selected genotoxic impurities in pharmaceutical raw materials and final products

Device

The fluorimetric measurements were carried out using a Jasco model FP-8300 spectrofluorimeter (Tokyo, Japan) equipped with a 1 cm quartz cuvette. Excitation and emission slit widths were adjusted to 5 nm. Spectra Manager software (Jasco Co., Tokyo, Japan) was applied for data processing and acquisition.

The pH was measured and adjusted using a pH meter, Jenway 3510 (UK).

A thermostatically controlled water bath, SY-2L4H (China), a benchtop centrifuge, Cyan-CL008 (Belgium) and an ultrasonic cleaner, SB-120DT (China) were also used.

Materials

Reference samples

2-AP (99.0%) and 3-AP (99.0%) were purchased from Sigma Aldrich (St. Louis, USA).

PX of purity (99.7%) was kindly provided by EL-Obour Modern Pharmaceutical Industries Company (El-Obour City, Egypt), TX of purity (100.1%) was kindly provided by EIPICO Company (Cairo, Egypt), purity ALO (99.7%) was kindly provided by Global Napi Pharmaceutical Company (October 6, Egypt), purity LIN (99.8%) was kindly provided by EVA Pharma Company (Cairo, Egypt ).

Pharmaceutical preparations

Different dosage forms were purchased in the local market. The composition of each was as follows:

Brexin tablets (product of Chiesi Pharma Company): labeled to contain (20 mg PX/tablet).

Feldene capsules, ampoules and gel (products of Pfizer Pharmaceutical Company) labeled to contain (20 mg PX/capsule and ampoule, 0.5% PX gel).

Dispercam suppositories (product of Medical Union Pharmaceuticals): labeled to contain (20 mg PX/suppository).

Epicotil (EIPICO Company products) tablets, bottles and suppositories labeled to contain (20 mg TX/tablet, bottle and suppository).

Oral capsules (product of Global Napi Pharmaceutical Company) labeled to contain (20 mg TX/capsule).

Chemical products

Sulfuric acid (H2SO4) (98%) was obtained from Piochem (Cairo, Egypt).

Sodium dihydrogen phosphate, phosphoric acid, methanol, ethanol and acetonitrile were purchased from Merck (Darmstadt, Germany).

Sodium dodecyl sulfate (SDS), Tween 20, Tween 80 and span 60 were purchased from Sigma Aldrich (St. Louis, USA).

Deionized water was used throughout the experiment.

Preparation of stock solutions and buffer solution

A stock standard solution equivalent to 100 µg/mL each of 2-AP and 3-AP was prepared separately by transferring 10.0 mg of 2-AP and 3-AP accurately weighed into a 100 mL volumetric flask and completed up to the mark with demineralised water. Further dilution was performed with the same solvent to obtain working standard solutions with a concentration of 1.00 µg/mL.

A 100 mM phosphate buffer (pH 3.0) was prepared by dissolving 12.0 g of monosodium phosphate in 800 mL of deionized water, the pH was adjusted with phosphoric acid, then diluted to 1000 mL using the same solvent22.

Construction of calibration curves

For 2-AP, into a series of 10 mL volumetric flasks, appropriate aliquots of the working standard solution covering the concentration range of 2.50 to 100 ng/mL were added and mixed with 1.0 mL of 100 mM phosphate buffer pH 3.0 (to obtain a final buffer concentration of 10 mM), then topped up to the mark with deionized water to obtain a final concentration range of 2.50 to 100 ng/mL.

For 3-AP, the same procedure was followed, except that 100 mM H2SO4 was used instead of phosphate buffer.

Fluorescence intensity was recorded at (λem370nm/λex298 nm) for 2-AP and (λem403nm/λex250 nm) for 3-AP. Standard curves were obtained by plotting fluorescence intensity versus analyte concentration (ng/mL) and corresponding regression equations were derived.

Dosing procedures for active pharmaceutical ingredients

Precisely weighed 10.0 mg of PX and TX and 100.0 mg of LIN and ALO were separately transferred into a series of 100 mL volumetric flasks. Dosed with appropriate aliquots of 2-AP (for PX and TX) and 3-AP (for ALO and LIN) to obtain final 2-AP and 3-AP concentrations of 2.50 to 100 ng/mL. The mixture was dissolved in 10 ml of methanol then sonicated for 5 min, the volume was made up to the mark with deionized water, then further diluted with deionized water to obtain a final concentration of 10 μg/mL of PX and TX, 100 μg/mL ALO and LIN. Then the procedure described under Construction of the calibration curve was played.

Dosage procedures for dosage forms

All dosage forms were spiked with appropriate aliquots of 2-AP and 3-AP working standard solutions to achieve final concentrations of 2.50-100 ng/mL. The final PX and TX concentration of each dosage form was adjusted to 10 μg/mL. Then the procedure described under Construction of the calibration curve was played.

Procedures for tablets

Ten Brexin tablets and Epicotil tablets were finely powdered separately and thoroughly mixed. An accurately weighed amount equivalent to 10 mg of PX and TX was separately transferred to a series of 100 mL volumetric flasks and dissolved in 10 mL of methanol, and spiked with increasing concentrations of 2-AP, the flasks were sonicated for 5 min and made up to volume with deionized water. The resulting solutions were filtered and further diluted with deionized water.

Procedures for capsules

The medicinal contents of ten Feldene capsules and Soral capsules were separately emptied, weighed, mixed. An accurately weighed amount equivalent to 10 mg of PX and TX was transferred separately into a series of 100 mL volumetric flasks and the procedure was completed as mentioned under Tablets.

Procedures for ampoules and vials

The contents of ten Feldene ampoules were mixed and a volume equivalent to 20 mg of PX was transferred to a series of 100 ml volumetric flasks, then spiked with increasing concentrations of 2-AP and made up to volume with water demineralised. The solutions obtained were then diluted with demineralised water.

Ten vials of Epicotil were reconstituted with deionized water, mixed thoroughly and transferred to a series of 100 ml volumetric flasks, the vials were washed with deionized water and the wash was transferred to the same vials and spiked with increasing concentrations of 2-AP, then made up to volume with deionized water. The solutions obtained were then diluted with demineralized water.

Freezing Procedures

An amount of Feldene gel equivalent to 10 mg of PX was weighed into a beaker, spiked with increasing concentrations of 2-AP and allowed to dissolve in 25 mL of ethanol and 2.0 mL of NaOH solution, after which, the solutions were filtered and transferred to a series of 100 mL volumetric flasks and made up to volume with deionized water. The solutions obtained were then diluted with demineralized water.

Procedures for suppositories

Accurately weighed amounts of Dispercam suppositories and Epicotil suppositories equivalent to 10 mg of PX and TX were placed separately in a beaker, added with increasing concentrations of 2-AP, and melted in a thermostatically controlled water bath set at 50–60 ° C, then, 5.0 ml of methanol was added and then heated again with gentle stirring, then the solutions were transferred to a series of 100 ml volumetric flasks and made up to volume with deionized water, the solutions were then centrifuged, filtered and further diluted with deionized water.

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