A new spectrofluorimetric method for the determination of ascorbic acid with bromocresol purple in pharmaceutical samples

Based on the interaction between ascorbic acid and bromocresol purple, a new simple, straightforward, and quick method for the quantification of ascorbic acid is proposed. The procedure is based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of bromocresol purple in the reaction between ascorbic acid and bromocresol purple in phosphate buffer solution (pH 6). The reduction of bromocresol purple fluorescence intensity is detected at 641 nm, while excitation occurs at 318 nm. The linear relationship between the reduced fluorescence intensity of bromocresol purple and the concentration of ascorbic acid is in the range 4.65 × 10–5 to 4.65 × 10–6 mol L−1 (R2 = 0.9964), with the detection limit of 8.77 × 10–7 mol L−1 and quantification limit of 2.35 × 10–5 mol L−1. The findings in this study further show that the new method provides good precision and repeatability, as well as satisfactory recovery values in terms of accuracy. The new method is tested on fifteen samples with different amounts of ascorbic acid and additional components. The effects of interfering components such as citrus bioflavonoids, citric acid, folic acid, paracetamol, calcium, and magnesium carbonate on the intensity of fluorescence of bromocresol purple are also investigated. The effects of interfering components such as citrus bioflavonoids (routine and hesperidin), citric acid, folic acid, paracetamol, calcium, and magnesium carbonate on the intensity of fluorescence of bromocresol purple are also investigated. The results of iodometric titration point out that the new method is effective for the determination of ascorbic acid in pharmaceutical samples. A new spectrofluorimetric method for determination of ascorbic acid in pharmaceutical samples using bromocresol purple. Determination of optimal parameters for ascorbic acid determination in a variety of pharmaceutical samples. Examination of the influence of additional substances in the pharmaceutical samples on the analysis. A new spectrofluorimetric method for determination of ascorbic acid in pharmaceutical samples using bromocresol purple. Determination of optimal parameters for ascorbic acid determination in a variety of pharmaceutical samples. Examination of the influence of additional substances in the pharmaceutical samples on the analysis.


Introduction
Ascorbic acid (AA) called vitamin C is a six-carbon lactone that is synthesized withinside the liver of most mammalian species from glucose. Humans, non-human primates, and guinea pigs don't posess the enzyme gulonolactone oxidase that is crucial for the synthesis of 2-keto-l-gulonolactone, the instant precursor of ascorbic acid. As an effect of now no longer taking nutrition C thru diet, a deficiency state happens which manifests with a huge spectrum of demonstrates that ascorbic acid can stifle the system's reactivity. As a result, this phenomenon is utilized to determine ascorbic acid in a range of materials [16].
This paper describe new, simple, sensitive, and rapid spectrofluorimetric method for determination of ascorbic acid in pharmaceutical samples. Bromocresol purple (BCP) was chosen as the chromogenic reagent for the determination of AA in pharmaceutical samples. The effects of various reaction conditions, such as acidity, BCP concentration, reaction time, instrumental parameters, as well as the influence of possible interfearing substances were carefully investigated. To the best of our knowledge, there has been no report on the application of bromocresol purple for the determination of AA by a spectrofluorimetric method.

Instrumentation
All fluorescence measurements were carried out on a lambda LS 55 luminescence spectrometer (Perkin-Elmer, Buckinghamshire, UK) equipped with a xenon lamp and a 1 cm quartz cell. pH adjustments were done on a digital pH meter Metler Toledo pH meter SG2 SevenGo.

Reagents and solutions used for spectrophotometric determination
All reagents used were analytical grade. A stock solution of ascorbic acid (Gram-mol, Croatia) 4.65 × 10 -4 mol L −1 is always prepared fresh due to the instability of AA by dissolving 0.0041 g of AA in a 50.0 mL calibrated flask and fill up to the mark with ultra-pure water (conductivity was: 0.055 S cm −1 , expressed as the resistivity of 18.2 MΩ cm −1 and TOC was 0.02 ppb). A stock solution of bromocresol purple (BCP) (Coleman & Bell Co, Norway) 1 × 10 -4 mol L −1 is prepared by dissolving 0.0054 g of BCP in a 100.0 mL graduated flask and filled up to the mark with ultra-pure water. All further dilutions are made with ultra-pure water. The BCP working concentration was 1 × 10 -5 mol L −1 . Phosphate buffer, pH = 6.4 was prepared by mixing 27.0 mL of dipotassium hydrogen phosphate (Merck, Germany) in a concentration of 1 mol L −1 and 72.2 mL of potassium dihydrogen phosphate (Semikem, Bosnia and Herzegovina) also at a concentration of 1 mol L −1 . The pH is adjusted with 2.0 mol L −1 sodium hydroxide. Pharmaceutical samples (tablets, powders, and effervescent tablets) used for analysis (15 samples) were commercially available. The solutions of pharmaceutical preparations were prepared by dissolving suitable amounts of | https://doi.org/10.1007/s42452-021-04820-0 Research Article the samples in a 50.0 mL calibrated flask and diluting to the mark with ultra-pure water.
The following commercial pharmaceutical samples in the form of tablets and powders have been analyzed by the proposed method. Table 1 shows pharmaceutical samples that were analyzed with the proposed method together with the accompanying components that can be found in tablets.
For the validation experiments, a 0.01 mol L −1 iodine solution was prepared and standardized according to the literature. The iodine method is recommended by the European Pharmacopoeia [23] as the standard method for the determination of AA in pharmaceutical preparations.
Possible interfering substances such as rutin, hesperidin, calcium carbonate, magnesium carbonate, citric acid, paracetamol, folic acid, zinc chloride, and iron(II) sulfate were examined. The influence of possible interfering substances on the relative intensity of the fluorescence signal was examined by analyzing the standard solution of AA and three different concentrations of the interfering substances. Concentrations of interfering substances were higher, the lower and same order of magnitude as the concentration of AA.

General procedure for the spectrofluorimetric quantification of ascorbic acid
An aliquot of AA (2 mL) of different dilutions, BCP (2 mL), and phosphate buffer (1 mL) were mixed in a 10.0 mL calibration flask. The volumetric flasks with different AA dilutions are then incubated at 40 °C for 15 min in a water bath, then left to cool to room temperature for 5 min. The final mixture was diluted to 10.0 mL with ultra-pure water. After cooling for 10 min, the relative fluorescence intensity signal was measured at working excitation and emission wavelengths (318 nm and 641 nm) with a 10.0 mm slit width of all open filters and at 1% of transmittance (1%T). The fluorescence intensity was also measured after 30 min under the above-mentioned conditions.

Results
This paper informs on a new method for the determination of ascorbic acid with bromocresol purple in pharmaceutical samples that was developed. Ascorbic acid in reaction with BCP is oxidized to dehydroascorbic acid (DHAA) and BCP is reduced to his reduced (Leuko-BCP) form in phosphate buffer pH = 6.4 (Fig. 1). The effect of decreasing relative fluorescence intensity was used for the estimation of AA content in investigated samples.

Determination of wavelength for a maximum of excitation and emission
Excitation and emission spectra of the reagent (phosphate buffer/BCP) without AA were scanned in the wavelength range of 200-800 nm for excitation and 200-900 nm for emission spectra using 10.0 mm slit widths and scan rates of 500 nm/min for both spectra. Excitation peaks were observed at 318, 381, 509 nm, and emission peaks at 320, 361, 641 nm. Then, excitation and emission spectra of the reagent with an added analyte, AA (4.65 × 10 -4 mol L −1 ) were scanned and a decrease of intensity of fluorescence (IF) values was observed on all peaks, particularly from 895.83 to 578.75 at 318 nm excitation wavelength, and from 1000.07 to 826.60 at 641 nm emission wavelength, and these were chosen to be the working wavelengths for this method (Fig. 2).

Optimization of experimental condition for the proposed method
To optimize the proposed method, the effects of various reaction conditions, such as pH, BCP concentration, reaction time, instrumental parameters (slits, open filters, and 1%T) were carefully investigated. Variables were changed in the experiments one by one, while others were kept constant. Furthermore, various validation parameters such as accuracy, precision, repeatability, etc. were also investigated and calculated to prove the validity of results provided by this method.

Influence of pH
The spectrophotometric measurement of AA with bromocresol purple in acidic conditions at pH = 3,0 was reported by Elgailani and Alghamdi [24]. However, this was not shown to be applicable for the spectrofluorimetric determination of AA. As a result, the acidity/basicity range in which the reaction occurs was tested between 3 and 8. Between pH 5 and 7, the best results were obtained. So the working pH of the phosphate buffer was further examined by 0.2 pH units in the range of 5 and 7. According to the literature data for electroanalysis of ascorbic acid using poly(bromocresol purple) [25] pH, 6.4 was the best. Our result also showed that the best reproducibly was achieved at pH 6.4 and this pH was taken for further experiments.

Influence of the concentrations of BCP
The effect of the concentration of BCP on the relative fluorescence intensity was examined in the concentration range of BCP from 1 × 10 -7 to 1 × 10 -4 mol L −1 .  highest IF was found to be at the concentration value of 1 × 10 -5 mol L −1 , (Fig. 3) whereas higher concentrations showed lower IFs, and with lower concentrations, it was not possible to construct the calibration curve.

Calibration Curve, LOD and LOQ
Under optimal conditions, the calibration curve was found to be linear in the concentration range of AA from 4.65 × 10 -5 to 4.65 × 10 -6 mol L −1 with a determination coefficient of R 2 = 0.9964, and the linear equation y = −61.836x + 8.5902. and recorded at 1%T. The linearity area is narrow, which is also a limitation of the method. The limit of detection was (LOD) 8.77 × 10 -7 mol L −1 , (calculated as 3σ) and limit of quantification (LOQ) 2.35 × 10 -5 mol L −1 , (calculated as 10σ) were determined using nine (n = 9) standard solutions of AA of the same concentration (2.48 × 10 -5 mol L −1 ), by measuring their IFs. Measurement precision (expressed as RSD) was found to be 8.46% for this concentration.
The calibration curve was also recorded with the instrument parameter all filters opened. It was found to be linear in the concentration range from 4.77 × 10 -5 to 4.77 × 10 -6 mol L −1 with a determination coefficient of R 2 = 0.9809, and the linear equation y−6 × 10 6 + 981.28 under the optimal condition Table 2.

Reaction time
The stability of the IF signal of Leuko-BCP concerning time was determined by measuring the reaction progression of an AA solution (4.65 × 10 -4 mol L −1 ) with the reagent for  13 min, and it was shown that the signal is stable 9 min after the reaction was initiated.

Accuracy, precision, and repeatability
The accuracy of this spectrofluorimetric method was assessed by analyzing AA solutions in the concentration range from 0.0480 mmol L −1 to 0.0058 mmol L −1 (n = 10 for each concentration) and by calculating SD, %RSD, and Recovery values (Table 3), which showed satisfactory accuracy.
To estimate precision and repeatability, the IF of one newly-prepared AA solution 0.477 mmol L −1 was measured periodically, in four replicates with n = 10 ( Table 4). Obtained results were taken for calculating SD and %RSD.

Sample preparation
The samples used for analysis were in different forms, with a different ascorbic acid content, and commercially available. Pharmaceutical samples (tablets, effervescent tablets, and one powdery form) were first weighed, then powdered in a mortar (except the powdery form of AA, sample). A suitable amount of powdered sample was dissolved in ultra-pure water in a 50.0 mL calibrated flask, which was the stock solution. Then further dilutions (1:10, 1:20, 1:100) were prepared with ultra-pure water in 10.0 mL calibrated flasks, which were the working sample solutions used for analysis. Simultaneously, the stock solution aliquots of   2.0 mL were centrifuged at 5000 rpm for 10 min at 20 °C. After the samples were centrifuged, identical dilutions were made with ultra-pure water as with the non-centrifuged stock solution to prepare working solutions. The samples were further treated according to the procedure and optimized conditions pH, the concentration of BCP reagent, time reaction, and instrumental parameters. Samples were measured in different concentrations (1:10, 1:20, 1:100), centrifugated and non-centrifugated, immediately after cooling and 30 min after, and with all filters open and 1%T. The results were calculated from a two-calibration curve using AA as a standard. Results are presented in Table 5. Samples containing zinc (samples 1 and 2) must be centrifuged and analyzed 30 min after the reaction. Sample 1 contains fluorescent riboflavin and it requires analysis with 1% T. The dilution corresponding to this type of sample is 1:10. Samples 3 (containing iron(II) lactate), sample 4 (containing paracetamol), and sample 5 (also containing other vitamins-multivitamin) require to be centrifuged, diluted 1:10, and analyzed after 30 min with 1% T. Sample 6 requires to be centrifuged because it contains cellulose and talc which can interfere with the analysis if not precipitated. It requires a 1:20 dilution and is analyzed with 1% T. Samples 8 and 9 can be analyzed without centrifugation immediately after the completing of the reaction. Sample 4 was diluted 1:10 and analyzed with all filters opened. Samples 5 and 9 are multivitamins, but sample 5 requires centrifugation because it contains inulin which is not well soluble in water, i.e., its solubility changes with temperature [26,27]. The difference in the analysis is that sample 5, which is multivitamin and contains a small amount of ascorbic acid (40 mg/tablet) should be diluted in a ratio of 1:20 and analyzed with 1% T due to the influence of other vitamins present in the sample. Samples 10 and 11 must be centrifuged before analysis because they contain inulin and talc which may limit the analysis especially in sample 11 where the AA content is lower. Also, both samples are diluted at 1:10. Sample 6 was also diluted at 1:20, centrifuged, and 30 min after the reaction was complete with all filters open. A sample of vitamin C containing 20 mg or less of bioflavonoids and 40 times more ascorbic acid was analyzed at a 1:10 dilution and in all filters open. Samples containing more than 20 mg of bioflavonoids must be diluted at 1:20 and worked with 1% T. The ascorbic acid content is 20 times higher than the bioflavonoid content.
Sample 15 could not be analyzed by the proposed method, even after centrifugation and dilution. It is believed that in addition to containing microcrystalline cellulose and magnesium stearate, it also contains croscarmellose sodium, which is cross-linked carboxymethyl cellulose. The crosslinking of carboxymethyl cellulose reduces the solubility in water, and at the same time allows the material to swell and absorb multiple weights in water. As a result, it provides superior drug dissolution and disintegration characteristics, thus improving the bioavailability of the formulas by bringing the active ingredients into better contact with body fluids. Its purpose in most pills, including dietary supplements, is to help the pill break down quickly in the gastrointestinal tract. If the tablet disintegrant is not included, the tablet may break down too slowly, in the wrong part of the gut, thus reducing the effectiveness and bioavailability of the active ingredients. Samples containing talc, inulin, cellulose, magnesium salts of fatty acids (in the form of magnesium stearate) require centrifugation before analysis. Samples containing hydroxypropylmethylcellulose do not require centrifugation because this form of cellulose is soluble in water. Multivitamin samples must be analyzed at 1% T. Samples containing zinc, iron, paracetamol should be diluted 1:10, centrifuged, and analyzed at 1%T.
Examination of the influence of individual interferences that can be found in pharmaceutical samples confirmed that additional components that are in addition to ascorbic acid must be taken into account because the preparation of the sample and the analysis itself depend on it.
Results obtained with the proposed method show good agreement with those obtained by the titrimetric method and the amount of AA stated on the declarations provide by the manufacturers, of simpler composition.
The content of AA for the sample that contains bioflavonoids calculated as hesperidin (Bio-C), samples 13 and 14, was higher than the result obtained by the titrimetric method. For sample 4 that contained paracetamol and some additional components, the content of AA measured with the proposed method is in agreement with the content of AA according to the labeled value. The presence of Zn in the investigated sample showed an impact on the relative fluorescence intensity. The AA content results in samples (samples 1 and 2) were higher than that obtained by titration.
Based on this data, the influence of bioflavonoids, metals such as zinc, iron, calcium, and magnesium in form of their salts, paracetamol, and folic acid on the relative fluorescence intensity were examined (Table 6).

Interferences
Rutin and hesperidin as citrus bioflavonoides, calcium carbonate, magnesium carbonate, citric acid, paracetamol, zinc chloride, folic acid, and iron(II) sulfate were investigated as possible interfering agents. By evaluating standard solutions of AA at one concentration of three different concentrations of the interfering substances, the effect of probable interfering substances on the relative intensity of the fluorescence signal of bromocresol purple was investigated. The concentrations of possible interfering substances were the same, higher, or lower than AA concentrations ( Table 6). All of these chemicals can be detected as ingredients or other components in pharmaceutical samples alongside AA. The concentrations of most possible interfering substances were greater than those present in pharmaceutical samples.
From the results presented in Table 6, it is clear that interfering substances like bioflavonoids or metals could interfere with the fluorescence intensity and give the wrong results on the quantity of AA. Paracetamol, folic acid, bioflavonoids show to have a major impact on signal intensity. Other investigated substances were investigated in a higher concentration than those which are actually present in the sample of AA. The interfering activity of magnesium carbonate, or some other type of magnesium salts like magnesium salts of fatty acids, calcium carbonate could be prevented by centrifugation or by diluting the sample unlike bioflavonoids, Zn, Fe, etc., which required some other different treatments.

Discussion
The purpose of this paper is to develop a new spectrofluorimetric method for the determination of ascorbic acid in pharmaceutical samples because spectrofluorimetry is a simple, low-cost, sensitive method that enables the measurement of low concentrations of the test analyte. After optimizing the method, which required testing different conditions under which the reaction between AA and BCP takes place, the goal was to test the method on as many pharmaceutical preparations containing various additional components besides AA. To achieve the goal, the method was tested on fifteen different samples having different AA content as well as additional components including bioflavonoids, iron, zinc, cellulose, citric acid, folic acid, paracetamol, etc. All samples were tested without prior preparation as well. with prior preparation involving centrifugation. Samples were analyzed with 1% T or with all filters open, as well as immediately after incubation and 30 min after incubation. Also, samples were prepared in three different dilutions to reduce the effect of interference, which was also subsequently examined. Since there is no universal method for all types of samples, this method also has its limitations and requirements related to sample preparation and analysis. Linearity area is narrow, which represents a limitation of this method and requires that samples be prepared in such a narrow concentration range. Samples that contain iron, zinc, paracetamol, magnesium salts like magnesium salts of fatty acids, cellulose, talc, inulin demand to be centrifugated before analysis. In samples that contain hydroxypropylmethylcellulose, it is not necessary to be centrifugated because this form of cellulose is soluble in water. Samples that contain bioflavonoids and riboflavin must be analyzed with 1%T because these components tend to fluoresce. Multivitamin samples must be analyzed at 1% T. Finally, samples that have a smaller amount of AA, and a lot of additional components are required to be diluted in ratio 1:20 so the influence of additional components can be reduced. Table 7 compares performance characteristics such as linearity range and limit of detection reported spectrofluorimetric methods. It is visible that some of the methods have a narrow linear range while others have a wider. Comparing the proposed method with other methods in the literature in terms of the field of linearity [14,28,29] the proposed method does not have a wide field of  Table 2, which is also its biggest limitation. The detection limit Table 7 is also not better than few other proposed methods [14-16, 21, 29, 31] for the determination of AA in pharmaceutical samples. However, this method is promising for the simple and relative rapid determination of AA. It can be applied to many pharmaceutical samples containing various additional components..

Conclusions
For the quantification of ascorbic acid, a variety of spectrophotometric and spectrofluorimetric methods are routinely utilized. They're utilized in the regular analysis since they're typically simple, operative, quick, cheap, and sensitive. Because there is no single method for all types of samples, each documented method, including this one, finds use for a different type of sample, which necessitates a different type of sample, which demands a new form of sample preparation. The proposed approach is simple to use and has a high sensitivity. Except for samples that include interfering compounds that must be eliminated before analysis, sample preparation is simple. Depending on the additional components present in the sample, the sample will be processed and evaluated differently. The method has the benefit of being able to be used on a large number of pharmaceutical samples. The method showed good precision and repeatability as well as satisfactory recovery values in terms of accuracy. A limitation of the method is the narrow linearity range. To our knowledge, there are no other reports on the spectrofluorimetric method for estimating AA content with bromocresole purple.
Further research could include applying this method to the real samples or trying to add some components or metal ions that could affect on the reaction to achieve better reproducibility, reaction rate, better linearity range, and maybe reduce sample preparation.
Author's contribution All authors have participated in this research, either by conception and design or analysis and interpretation of the data; drafting the article, or revising it critically for important intellectual content.
Availability of data and material On the request from the corresponding author.
Code availability Not applicable.

Conflict of interest
The authors declare that they have no conflict of interest.
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