All chemicals were of reagent grade and were used without further purification. Citric acid samples were obtained from two different sources - Merck (Darmstadt, Germany) and BDH Chem (Poole, England). 8-Hydroxyquinoline was purchased from two manufacturers - Sigma-aldrich (St. Louis, MO) and Fluka (Munich, Germany). Chloroform (RCI Labscan, Thailand) and distilled water was used as solvents.

The effects of the matrix, which is produced as a result of the presence of citric acid, was examined by comparing the standard curves of aluminum solutions without citric acid, with those in which citric acid was present. For this purpose, aluminum standard solutions in the concentration range of 0–0.06 mg/ml, prepared using water and 0.2 g/ml citric acid solution as solvent were used. After the addition of acetate buffer (pH 6.0), the solutions were extracted with three successive portions of 0.5% w/v 8-hydroxyquinoline in chloroform, following the procedure described in the USP. The extracts from the chloroform phase were then subjected to fluorescence measurement, setting the excitation wavelength and emission wavelength at 492 and 518 nm, respectively. To obtain the F.I. data of the reagent blanks, chloroform was used to zero the instrument. The analyses were done in triplicate.

To ensure the reliability and reproducibility of the results, the analyses were carried out using citric acid obtained from two different sources, 8-hydroxyquinoline from two manufacturers, and two spectrofluorometer models i.e. RF-6000 and RF-1501 by Shimadzu (Japan). It should be noted that the instrument settings of the two spectrofluorometers were different. For RF-6000, the excitation and emission bandwidths were 3 nm and 3 nm, respectively, and the “high” sensitivity was set. For RF-1501, the excitation and emission bandwidths were 10 nm and 10 nm, respectively, and the “high” sensitivity was set. The contents of aluminum in the citric acid sourced from Merck and BDH Chem were 0.033 and 0.048 ppm, respectively, as determined by inductively coupled plasma-mass spectrometry (ICP-MS).

The concentration of aluminum in the citric acid as well as in the aqueous phase before and after extraction was determined using an ICP-MS spectrometer (Model 7500ce, Agilent). Collision/reaction cells were adopted for removing spectral interferences in ICP-MS. The operating conditions were an ICP RF power of 1500 W, an ultrapure grade carrier (argon, 99.9995% pure) flow rate of 1.5 l/min, and a nebulizer pump speed of 0.1 rps. The signal intensities were compared to a calibration curve of aluminum which was prepared in the range of 0.5–100 mg/l. The data are presented as the average of triplicate determinations.

Prior to extraction, the pH of the aluminum solutions was measured. Even with the aid of a buffer added, the pH of the solution containing citric acid (pH 2.2) was very different from that in the water (pH 5.7). Once 8-hydroxyquinoline in chloroform was added and the solutions were shaken, the upper aqueous phase of the extraction of aluminum in the citric acid solution appeared to be more slightly yellow than that of the solution in the water. Since 8-hydroxyquinoline is insoluble in water, but soluble in chloroform and acidic aqueous medium (Marczenko and Balcerzak 2000), the low pH of the citric acid solution is likely to have promoted an increased distribution of 8-hydroxyquinoline into the aqueous phase, as seen by the more yellow color which it imparted, compared to the solution that was prepared in water.

Over the range (0–0.06 mg/ml) that covered the concentration of the aluminum standard solution mentioned in the compendial method (0.04 mg/ml), the standard curves obtained from the aluminum solutions prepared in 0.2 g/ml citric acid solution and those prepared in water, differed greatly in terms of both slope and y-intercept (Figure 1), indicating the interference effects caused by citric acid. In addition, the F.I. values on the plot belonging to the citric acid solution were much lower than those of the water. Conducting the experiments using chemicals and instruments from different manufacturers produced the same results. In another experiment, in which varying concentrations of citric acid were added to 0.04 mg/ml aluminum solutions, a decrease in the F.I. was clearly seen when the co-existing concentration of the citric acid increased (Figure 2). From these findings, it can be concluded that the sample matrix, i.e. citric acid, interfered with the test by dramatically lowering the analytical signals. Consequently, the decision of acceptance, which is made by comparing the F.I. of a sample solution with that of a standard solution, as described in the pharmacopeias, can be seen to be misleading and result in the underestimation of the aluminum content in samples.

Figure 1. 

Standard curves of aluminum in water and in citric acid solutions, prepared using chemicals and instruments of different manufacturers; (a) citric acid of Merck/8-hydroxyquinoline of Sigma-aldrich/ spectrofluorometer RF-1501, (b) citric acid of BDH Chem/8-hydroxyquinoline of Sigma-aldrich/spectrofluorometer RF-1501, (c) citric acid of Merck/8-hydroxyquinoline of Fluka/spectrofluorometer RF-1501, (d) citric acid of Merck/8-hydroxyquinoline of Sigma-aldrich/spectrofluorometer RF-6000.

Figure 2. 

Effect of the concentration of citric acid co-existing in 0.04 mg/ml aluminum solution on the fluorescence intensity of the extract as measured using RF-6000 spectrofluorometer (Shimadzu).

As previously mentioned, the pH of the aluminum solution containing citric acid was much lower than that in water. Therefore, it was investigated to determine whether the interference was attributable to the acidity of citric acid. By adjusting the pH of the citric acid solution from pH 2.2 to 4.0, the F.I. increased (Figure 3). However, further increasing the pH of the citric acid solution to 5.7 did not give an equal F.I. to that of the aluminum solution in water. These results imply that while the acidity of citric acid may affect the distribution of amphoteric 8-hydroxyquinoline and/or the extractability of the aluminum complex into chloroform which occurred to a greater extent at neutral pH (Marczenko and Balcerzak 2000), it was not the only reason for the interference.

Figure 3. 

Effect of pH of 0.04 mg/ml aluminum solution prepared in 0.2 g/ml citric acid solution, on the fluorescence intensity of the extract, compared with that of a solution prepared in water. The measurements were done using RF-6000 spectrofluorometer (Shimadzu).

Citric acid is capable of chelating metals including aluminum (Feng et al. 1990; Karbouj 2007; Asemave 2018). Therefore, it is possible that the citric acid which was present in the aqueous phase of the test competed with 8-hydroxyquinoline in reacting with the aluminum, resulting in the partition of aluminum species into the aqueous phase. As hypothesized, the quantitation of aluminum in the aqueous phases using ICP-MS revealed that following the extraction of aluminum in the citric acid solution, the majority of aluminum remained present in the aqueous phase (Figure 4), probably due to the formation of water-soluble complex with citric acid. In contrast, in the solution without citric acid, aluminum was almost completely extracted into the organic phase, since the complex of aluminum and 8-hydroxyquinoline is soluble in chloroform. Based on these results, it is evidenced that citric acid causes interference in the test owing to its acidity and metal-chelating capabilities.

Figure 4. 

Concentration of aluminum in the aqueous phase in the extraction of 0.04 mg/ml aluminum solution prepared in water and in the solution containing 0.2 g/ml citric acid. Phase.

In the view of the pharmaceutical quality control and manufacturing, the tests of active pharmaceutical ingredients which are affected by matrix interference may cause inaccurate conclusions, and it can become a safety hazard for consumers. Consequently, the revision of the test and/or the investigation of more reliable assays which overcome the matrix effects are recommended. For this purpose, the limit test based on atomic absorption spectroscopy as described in some pharmacopeias for the determination of aluminum in certain raw materials e.g. potassium chloride, sodium acetate and sodium carbonate (The United States Pharmacopeial Convention 2021), or those reported in some research articles (Woolfson and Gracey 1988; Sharma et al. 2015) may be used. Alternatively, selective and sensitive assays relying on atomic emission spectroscopy such as ICP-MS (Li et al. 2017) as well as spectrophotometry (Ahmed and Hossan 1995; Kamino et al. 2005) and fluorimetry (Raggi et al. 1999; Hossain et al. 2020) previously proposed for the determination of aluminum in pharmaceutical and biological samples may be interesting choices. Prior to use for the replacement of the current compendial test, the method validation and investigation of matrix interference caused by citric acid must be carried out to ensure that reliable results will be obtained.