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Article

Determination of Zn2+ in Solid Pharmaceutical Dosage Forms by Means of Spectrophotometry in Micellar Media: Method Validation

by
Luca Liviu Rus
1,
Anca Maria Juncan
1,*,
Veronica Isabela Crăciun
1,
Adina Frum
1,
Simona-Codruța Hegheș
2,
Anca Butuca
1,
Carmen Maximiliana Dobrea
1,
Adriana Aurelia Chis
1,
Andrei Catalin Muntean
1,
Andreea Loredana Vonica-Ţincu
1 and
Claudiu Morgovan
1
1
Preclinical Department, Faculty of Medicine, “Lucian Blaga” University, 2A, Lucian Blaga Street, 550169 Sibiu, Romania
2
Department of Drug Analysis, “Iuliu Hațieganu” University of Medicine and Pharmacy, 6 Pasteur Street, 400349 Cluj-Napoca, Romania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(9), 4476; https://doi.org/10.3390/app12094476
Submission received: 22 March 2022 / Revised: 23 April 2022 / Accepted: 27 April 2022 / Published: 28 April 2022
(This article belongs to the Special Issue Analytical Techniques for the Determination of Bioactive Food Compounds)
Browse Figure
Versions Notes

Abstract

:
In this study, a spectrophotometric method for the quantitative determination of zinc in a dietary supplement was validated. Zinc forms a ternary complex with Xylenol Orange and cetylpyridinium chloride in acetate buffer at pH = 5.5. The complex exhibits an absorption maximum at 560 nm. Linearity (range was 0.5–1.5 µg/mL, and correlation coefficient of the calibration curve was 0.9998), precision (repeatability variation coefficient and reproducibility variation coefficient were 1.57% and 1.77%, respectively), and accuracy (confidence interval of the mean recovery was 98.29–100.55%) of this method fulfil current pharmacopeial requests. Results obtained by spectrophotometric method were statistically compared with ones obtained by atomic absorption spectrometry and good agreement was found. The method can be applied routinely for quality control of solid pharmaceutical dosage forms containing zinc.

1. Introduction

Oxidative stress can be associated with many pathologies [1,2]. Together with other compounds (vitamin C, vitamin E, thioctic acid, glutathione, etc.), zinc provides protection against oxidative damage [1,2,3].
Zinc is the second most abundant metal in the human body [4]. Practically, it is an oligo-element essential for human health because it is a component of proteins involved in cell structures [5] and on the other hand it is a cofactor for more than 300 enzymes involved in many processes, including the cellular respiration, immunity, DNA and protein synthesis, metabolism, cell division, etc. [3,4,5,6,7]. Thus, its deficiency can lead to serious damage, being responsible for different pathologies, for example, the acute deficiency decreases the innate and adaptive immunity and chronic deficiency increases the production of pro-inflammatory cytokines and leads to severe inflammatory diseases such as rheumatoid arthritis [5]. Obese patients showed alteration of zinc metabolism and that can lead to endocrine disorders. Zinc-α2-glycoprotein (Zn-2α-G) is an adipokine with lipid-mobilizing and anti-inflammatory activity. Thus, low levels of serum zinc and Zn-2α-G were identified in obese patients [8]. On the other hand, blood clotting disturbances are related to zinc intake: hypozincemia leads to poor aggregation of platelets and prolongation of bleeding time and, opposite, hyperzincemia leads to increased coagulability [9].
The zinc supplementation is efficient in the management of patients with type 2 diabetes mellitus (DM2), which exbibit hypozincemia (possibly due to reduced gastrointestinal absorption). It was observed that zinc supplementation in DM2 patients resulted in reduced glycated hemoglobin percentage [10]. Likewise, zinc supplementation was proved to reduce severity and duration of diarrhea in children because of intestinal mucosa regeneration and facilitation of electrolyte transport [11]. According to another study, zinc is linked to oxidative stress and may be important in prevention and treatment of sarcopenia [12]. Additionally, zinc proved to be effective in the treatment of acne vulgaris, in several clinical trials [13]. Active transport of zinc inside prostate cells is performed by a zinc transport protein (ZIP1). ZIP1 downregulation may be involved in the decrease in zinc inside malignant cells. Zinc treatment of human prostate tumors was demonstrated [14].
Nevertheless, zinc may exhibit several adverse events and may interact with quinolones and tetracycline (decreasing antibiotic absorption) [15]. On the other hand, using zinc in the treatment of neonatal hyperbilirubinemia is not encouraged [16]. Therefore, dietary supplements (DS) can be useful in treatment of several pathologies, but special attention must be paid to nutrivigilance issues [15].
Several zinc complexes with active pharmaceutical ingredients were synthetized and tested in order to improve biological properties of the parent molecules [17,18,19,20,21]. Thus, for improving their efficiency and reducing the risks, the quality control of DS is essential for their biopharmaceutical profile. Many papers were published describing quantitative determination of zinc in pharmaceutical and biological matrices: neutron activation analysis [22], AAS [23,24], voltammetry [25,26], differential-pulse polarography [27], spectrofluorimetric analysis [28], electrodialysis coupled with AAS [29], ion-selective electrode [30], capillary zone electrophoresis [31], solid phase photometry [32,33], derivative spectrophotometry [34,35,36,37], and 0 order spectrophotometry [37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54]. Nowadays pharmacopoeias recommend quantification on zinc in dietary supplements by means of atomic absorption spectrometry (AAS) [55]. The AAS method requires expensive equipment and relatively expensive reagents.
Zinc forms binary [56] and ternary [57] complexes with Xylenol Orange (XyO) in acidic buffered media. A ternary complex forms in micellar media with cetylpyridinium chloride (CtyPyChl) in the ratio 1:2:4 (Zn2+:XyO:CtyPyChl). This method was used for quantitative determination of zinc in an ointment [57].
Considering these, the aim of the present study is the development and validation of a cheap and fast spectrophotometric method for the determination of zinc (by means of a ternary complex with XyO) from a DS formulated as a solid pharmaceutical dosage form for oral administration, taking into consideration the latest validation guidelines and protocols [58,59,60,61].

2. Materials and Methods

2.1. Reagents for Spectrophotometric Method

Xylenol Orange (XyO) was produced by Sigma-Aldrich, cetylpyridinium chloride (CtyPyChl) was purchased from Merck, Chimopar supplied zinc sulphate heptahydrate, sodium acetate trihydrate, glacial acetic acid, sodium hydroxide, and hydrochloric acid 0.1 M. All reagents were analytical grade (purity > 99.5%), and in all experiments, ultrapure water was used (conductivity 0.05 µS/cm).

2.2. Reagents for Atomic Absorption Spectrometry Method

Multielement standard solution (zinc concentration 100 mg/L) and 37% hydrochloric acid were produced by Merck and potassium chloride was supplied by Sigma Aldrich. All reagents were analytical grade (purity > 99.5%), and in all experiments, ultrapure water was used (conductivity 0.05 µS/cm).

2.3. Raw Materials and Industrial Batches

A synthetic mixture of dietary supplement components (SMDSC) was prepared with calcium carbonate (Shanghai Nuocheng, Shanghai, China), magnesium oxide (Dr. Paul Lohmann, Emmerthal, Germany), mannitol (Roquette, Lestrem, France), pregelatinized corn starch (Colorcon, Harleysville, PA, USA), Kollidon 30 (Basf, Ludwigshafen, Germany), Aerosil 200 (Evonik Industries, Essen, Germany), zinc oxide (Dr. Paul Lohmann, Emmerthal, Germany), glyceryl dibehenate (Gattefossé, Saint-Priest, France), and cholecalciferol (DSM Nutritional Products Ltd., Sisseln, Switzerland) and four industrial batches of the DS “Calciu + Magneziu + Zinc + Vitamina D3” (Polisano Pharmaceuticals S.A., Sibiu, Romania) were used for method development.

2.4. Apparatus and Software

An atomic absorption spectrometer NovAA® 400 with integrated software from Analytik Jena, was equipped with zinc hallow cathode lamp (also from Analytik Jena, Germany) and was operated for the flame technique (acetylene–air). The used spectrophotometer was: (s1) UV-1900 spectrophotometer with UVProbe 2.7 software (Shimadzu, Kyoto, Japan). For robustness, a second spectrophotometer was used: (s2) Thermo Evolution 300 with VISION Software Suite (Thermo Fisher Scientific, Waltham, MA, USA). Weighing was performed on Mettler Toledo balances (ME104T/00 and ME2002T/00) and calcination of samples took place in a Naberthem furnace. pH readings were performed with SevenExcellence (Mettler Toledo, Columbus, OH, USA) multiparameter equipped with a glass electrode and for heating and sonication a LBS2 4.5L ultrasonic water bath from Falc Instruments was used.

2.5. Synthetic Mixture of Dietary Supplement Components Preparation

The SMDSC was prepared according to the notification no. 3083/25.07.2016 (issued by the Ministry of Health from Romania) for the dietary supplement “Calciu + Magneziu + Zinc + Vitamina D3” (notification holder Polisano Pharmaceuticals S.A., Sibiu, Romania). Appropriate amounts of raw materials were weighed on a three-decimal balance. The raw materials were mixed in a mortar with a polyethylene terephthalate card, for 20 min.

2.6. Calibration, Sample Preparation, and Spectra Recording for the Spectrophotometric Method

A series of stock solutions were prepared by weighing suitable amounts of zinc sulphate heptahydrate (A), XyO (B), and CtyPyChl (C). Concentrations of stock solutions were 10 mM for Zn2+, 1 mM for XyO, and 10 mM for CtyPyChl. Aliquots ranging from 20 to 60 μL (A) were transferred to 25 mL volumetric flasks. In each flask, 2 mL of stock B and 1 mL of stock C were transferred. Each flask was filled to the mark with acetate buffer (0.5 M, pH = 5.5). Suitable amounts of real samples (both SMDSC and industrial batches) were weighed and transferred to 250 mL volumetric flasks. In each flask, 100 mL of hydrochloric acid 0.1 M was added. Flasks were sonicated for 15 min at 30 °C and filled up to the mark with the same solvent. Solutions were filtered and aliquots of 2 mL were transferred in 50 mL volumetric flasks together with 4 mL of stock B and 2 mL of stock C.
Spectra of calibration solutions and real samples were recorded, in glass 1 cm cuvettes, considering a reagent blank, in the wavelength interval 400–600 nm.

2.7. Calibration and Sample Preparation for the AAS Method

From multielement standard solution (100 mg/L), 5 aliquots (ranging 100 to 1200 μL) were transferred to 100 mL volumetric flasks together with 100 mg of potassium chloride. Flasks were filled to the mark with hydrochloric acid 0.125 M. Blank solution was prepared from 100 mg of potassium chloride dissolved in hydrochloric acid 0.125 M, in a 100 mL volumetric flask. Suitable amounts of real samples (industrial batches) were transferred in porcelain crucibles and calcinated for at least 6 h at 550 °C. After cooling, 15 mL of 37% hydrochloric acid was added to each crucible heating slowly on the water bath for 30 min. The mix was transferred to a 100 mL volumetric flask and the flask was filled to the mark with purified water. Then, 800 μL of this solution was transferred to a 100 mL volumetric flask together with 100 mg of potassium chloride and the flask was filled to the mark with hydrochloric acid 0.125 M. Absorbances were measured at 213.9 nm using a zinc hallow cathode lamp and deuterium background.

2.8. Spectrophotometric Method Validation Protocol

2.8.1. Linearity

Linearity was tested at 5 concentration levels (k = 5) in triplicate for both standard and SMDSC. Linearity was tested on three different days. Calibration curves (3 for standard and 3 for SMDSC) were plotted by linear regression. The correlation coefficient (r) should be above 0.99. For linearity, the following statistical tests were performed: Cochrane test (for intragroup variances homogeneity testing), Fisher test (slope significance evaluation and regression curves validity), t Student test (intercept comparison with null). Furthermore, t Student tests were performed (regression curves slopes and intercepts comparison) for SMDSC. In all tests, a 5% error probability was considered for all k = 5 concentration levels.

2.8.2. Precision

In order to prove the precision of the proposed method, repeatability and reproducibility were evaluated. Determinations were performed on a single concentration level (100%), 6 replicates per day on three different days (a total of 18 samples). The following statistical tests/parameters were performed/calculated: intragroup variances homogeneity (Cochrane test), repeatability variation coefficient (CVr%), and reproducibility variation coefficient (CVR%). In all tests, a 5% error probability was considered for all 18 samples.

2.8.3. Accuracy

Accuracy was estimated in terms of recovery using a calibration curve (constructed as described in the linearity protocol). Statistical analysis of accuracy consisted of s Cochrane test (for intragroup variance homogeneity), Fisher test (medium recovery validity), and t Student test (confidence interval for mean recovery), considering a 5% error probability.

2.8.4. Limit of Detection and Quantification

Limit of detection (LOD) and limit of quantification (LOQ) are parameters related to analytical method sensitivity and were calculated based on Equation (1).
LOD = 3.3 σ/s and LOQ = 10 σ/s
where σ is the standard deviation of the intercept of the calibration curve and s is the slope of the calibration curve.

2.8.5. Robustness

For the study of robustness, two parameters were varied: (1) apparatus and (2) zinc to other components ratio. The calibration curves were determined on two spectrophotometers (s1 and s2). Two SMDSCs were prepared by using zinc levels of 80% and 120% considering the industrial formula as the 100% level. Six samples were analyzed for each level (80% and 120%).

2.9. Statistical Comparison of Spectrophotometric and AAS Results

Comparison of results generated by the proposed method and AAS method was performed by means of a paired t Student test, considering a 5% error probability.
All statistical tests described in the validation protocol and comparison of spectrophotometric and AAS results were performed using Microsoft Excel® (Office 365 personal edition).

3. Results

UV-Vis (400–600 nm) spectra of calibration standards from day 1 of the validation protocol are presented in Figure 1.
Spectra of the ternary complex formed Zn2+-XyO-CtyPyChl presents an absorption minimum (426.5 nm), an isosbestic point (476 nm), and an absorption maximum (560 nm). For calibration and sample analysis purposes the absorption maximum was used.

3.1. Spectrophotometric Method Validation Report

3.1.1. Linearity

The range was 0.5–1.5 μg/mL for the proposed method and 0.1–1.2 μg/mL for the AAS method. As described in Table 1, in both cases correlation coefficients (r) were above 0.99. Intragroup variances are homogeneous, both calculated values for etalon and SMDSC fulfilled the Cohrane test (C < Ctheor), calibration curves have significant slopes (F > Ftheor), and regression curves are valid (F < Ftheor). Intercepts significantly differ from 0 since in both cases calculated t is below critical t (t < ttheor).
Additionally, slopes and intercepts for standard and SMDSC do not differ significantly (t < ttheor). All statistical tests for linearity fulfill predefined acceptance criteria.

3.1.2. Precision

As stated in Table 2, intragroup variances are homogeneous (C < Ctheor) and both CVr% and CVR% are below 2%. The proposed method is precise.

3.1.3. Accuracy

As stated in Table 3, variances are homogeneous (C < Ctheor), medium recovery is valid (F < Ftheor), and the confidence interval is close to 100%. The method is accurate.

3.1.4. LOD and LOQ

For the proposed method, the calculated LOD and LOQ were 4.93 and 14.92 µg/L, respectively (s = 5.69 × 10−4).

3.1.5. Robustness

The results for robustness are presented in Table 4.

3.2. Statistical Comparison of Spectrophotometric and AAS Results

The results (obtained by both proposed and AAS method) for four industrial batches are presented in Table 5.
As stated in Table 5, the results obtained by applying the proposed method and the AAS method do not differ significantly (t < ttheor).

4. Discussion

Due to its important biological role, zinc is often used as a dietary supplement. In order to use safe and efficient products for improving the quality of patients’ lives, the manufacturer should improve the quality control process using reliable, robust, easy to apply, and inexpensive methods of analysis. Thus, several modern analytical techniques are used for zinc determination from pharmaceutical forms. These are complex and difficult to apply in situ. On the other hand, they require expensive laboratory equipment, specialized staff, and a long period of time for processing. Spectrophotometric methods have a series of advantages over other advanced techniques, such as: simplicity, speed, low cost and maintenance, portable instrumentation, etc. Additionally, these methods should be validated, because they represent real competitive techniques for quality control of dietary supplements, if the experimental conditions are rigorously controlled [62].
Table 6 presents a comparison of current analytical method characteristics with previously published 0 order spectrophotometric methods, for Zn2+ determination in solid pharmaceutical dosage forms.
As presented in Table 6, the proposed method has a comparable range with other published 0 order spectrophotometric methods. The novelty of the method we described consists of a better characterization of the ternary complex (absorption minimum and isosbestic point). Quantitative determination could be performed at minimum wavelength and the presence of an isosbestic point could be valuable for the development of methods for quantitation of a mixture of ions that can form complexes with Xylenol Orange, in the future.
The current work did not try to study the whole range for which Beer’s law was obeyed, but to cover the linearity interval 80–120%, as recommended by the ICH guideline. Several statistical tests were applied in order to perform a statistical evaluation of linearity (Cochrane, Fisher, and t-Student).
The correlation coefficient is higher than 0.999 in the case of all presented spectrophotometric methods. Despite some of the methods presented in Table 6, the current method does not involve digestion with concentrated acids, calcination, or liquid–liquid extraction with toxic solvents.
Additionally, the current method is quite sensitive, considering its LOD.
In terms of precision, the intragroup variances are homogeneous (Cochrane test was passed). Moreover, CVr% and CVR% were 1.57% and 1.77%, respectively.
The statistical evaluation of accuracy revealed that intragroup variances are homogeneous (Cochrane test was passed), medium recovery is valid (as Fisher test showed), and confidence interval of the mean recovery is narrow and very close to 100%.
For the robustness study, the two variated parameters were apparatus and zinc to other components ratio. The intercept and slope of the calibration curves determined by means of the two spectrophotometers are comparable. Additionally, the confidence intervals of the mean recovery regarding the analyzed zinc levels (80% and 120%) are comparable to the 100% one.
Finally, statistical comparison of the results obtained by means of the current method and AAS method revealed no differences between results.

5. Conclusions

A spectrophotometric method in micellar media was proposed for quality assessment of a dietary supplement containing zinc, calcium, magnesium, and vitamin D3. It is based on the complex formed by Zn2+ with Xylenol Orange, and cetylpyridinium chloride in acetate buffer (pH = 5.5). The validating terms were linearity, precision, and accuracy based on the latest protocols and guidelines. Tests were performed on real samples (industrial batches and synthetic mixtures of dietary supplement components). Additionally, real samples were analyzed by means of an atomic absorption spectrometric method and good agreement was found with the stated amount of zinc in the dietary supplement “Calciu + Magneziu + Zinc + Vitamina D3”. The validated method is a cheaper, precise, and accurate alternative to the AAS method (which requires expensive equipment and long analysis times).

Author Contributions

Conceptualization, L.L.R., V.I.C., S.-C.H. and A.L.V.-Ţ.; methodology, L.L.R. and A.F.; software, L.L.R., A.M.J. and C.M.; validation, L.L.R., S.-C.H. and C.M.D.; formal analysis, A.B., A.C.M. and L.L.R.; investigation, L.L.R. and A.L.V.-Ţ.; resources, A.A.C.; data curation, L.L.R., S.-C.H. and A.F.; writing—original draft preparation, L.L.R., A.B., V.I.C., A.M.J., A.L.V.-Ţ. and C.M.; writing—review and editing, L.L.R., A.F., A.A.C. and A.L.V.-Ţ.; visualization, L.L.R., V.I.C., A.M.J., A.F., S.-C.H., A.A.C., A.B., A.C.M., C.M., C.M.D. and A.L.V.-Ţ. supervision, L.L.R., C.M., A.F. and A.L.V.-Ţ. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors wish to thank Polisano Pharmaceuticals SA for its generous contributions of research facilities and resources.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Juncan, A.M.; Rus, L.L.; Gligor, F.G.; Morgovan, C. Determination of Alpha-Tocopherol Acetate in an Anti-Aging Cosmetic Cream by Gas Chromatography. Rev. Chim. 2019, 70, 555–559. [Google Scholar] [CrossRef]
  2. Ghibu, S.; Craciun, C.E.; Rusu, R.; Morgovan, C.; Mogosan, C.; Rochette, L.; Gal, A.F.; Dronca, M. Impact of Alpha-Lipoic Acid Chronic Discontinuous Treatment in Cardiometabolic Disorders and Oxidative Stress Induced by Fructose Intake in Rats. Antioxidants 2019, 8, 636. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Jarosz, M.; Olbert, M.; Wyszogrodzka, G.; Młyniec, K.; Librowski, T. Antioxidant and Anti-Inflammatory Effects of Zinc. Zinc-Dependent NF-ΚB Signaling. Inflammopharmacology 2017, 25, 11–24. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Mccall, K.A.; Huang, C.-C.; Fierke, C.A. Function and Mechanism of Zinc Metalloenzymes. J. Nutr. 2000, 130, 1437S–1446S. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Bonaventura, P.; Benedetti, G.; Albarède, F.; Miossec, P. Zinc and Its Role in Immunity and Inflammation. Autoimmun. Rev. 2015, 14, 277–285. [Google Scholar] [CrossRef]
  6. Lin, P.-H.; Sermersheim, M.; Li, H.; Lee, P.H.U.; Steinberg, S.M.; Ma, J. Zinc in Wound Healing Modulation. Nutrients 2018, 10, 16. [Google Scholar] [CrossRef] [Green Version]
  7. Rochette, L.; Ghibu, S. Mechanics Insights of Alpha-lipoic Acid against Cardiovascular Diseases during COVID-19 Infection. Int. J. Mol. Sci. 2021, 22, 7979. [Google Scholar] [CrossRef]
  8. Severo, J.S.; Morais, J.B.S.; Beserra, J.B.; dos Santos, L.R.; de Sousa Melo, S.R.; de Sousa, G.S.; de Matos Neto, E.M.; Henriques, G.S.; do Nascimento Marreiro, D. Role of Zinc in Zinc-α2-Glycoprotein Metabolism in Obesity: A Review of Literature. Biol. Trace Elem. Res. 2020, 193, 81–88. [Google Scholar] [CrossRef]
  9. Tubek, S.; Grzanka, P.; Tubek, I. Role of Zinc in Hemostasis: A Review. Biol. Trace Elem. Res. 2008, 121, 1–8. [Google Scholar] [CrossRef]
  10. de Carvalho, G.B.; Brandão-Lima, P.N.; Maia, C.S.C.; Barbosa, K.B.F.; Pires, L.V. Zinc’s Role in the Glycemic Control of Patients with Type 2 Diabetes: A Systematic Review. BioMetals 2017, 30, 151–162. [Google Scholar] [CrossRef]
  11. Putri, W.B.; Akhmad, S.A.; Desrini, S. The Role of Zinc Supplementation for Diarrhoea in Children: A Critical Review. Bangladesh J. Med. Sci. 2019, 18, 190–195. [Google Scholar] [CrossRef]
  12. van Dronkelaar, C.; van Velzen, A.; Abdelrazek, M.; van der Steen, A.; Weijs, P.J.M.; Tieland, M. Minerals and Sarcopenia; The Role of Calcium, Iron, Magnesium, Phosphorus, Potassium, Selenium, Sodium, and Zinc on Muscle Mass, Muscle Strength, and Physical Performance in Older Adults: A Systematic Review. J. Am. Med. Dir. Assoc. 2018, 19, 6–11.e3. [Google Scholar] [CrossRef] [PubMed]
  13. Cervantes, J.; Eber, A.E.; Perper, M.; Nascimento, V.M.; Nouri, K.; Keri, J.E. The Role of Zinc in the Treatment of Acne: A Review of the Literature. Dermatol. Ther. 2018, 31, e12576. [Google Scholar] [CrossRef] [PubMed]
  14. Costello, L.C.; Franklin, R.B. A Comprehensive Review of the Role of Zinc in Normal Prostate Function and Metabolism; and Its Implications in Prostate Cancer. Arch. Biochem. Biophys. 2016, 611, 100–112. [Google Scholar] [CrossRef] [Green Version]
  15. Morgovan, C.; Ghibu, S.; Maria Juncan, A.; Liviu Rus, L.; Butucă, A.; Vonica, L.; Muntean, A.; Moş, L.; Gligor, F.; Olah, N. Nutrivigilance: A New Activity in the Field of Dietary Supplements. Farmacia 2019, 67, 537–544. [Google Scholar] [CrossRef]
  16. Sharma, D.; Farahbakhsh, N.; Sharma, P.; Shastri, S. Role of Oral Zinc Supplementation for Reduction of Neonatal Hyperbilirubinemia: A Systematic Review of Current Evidence. J. Matern. Fetal. Neonatal. Med. 2017, 30, 1953–1962. [Google Scholar] [CrossRef]
  17. Tita, B.; Morgovan, C.; Tita, D.; Neag, T.A. Anti-Inflammatory Drugs Interacting with Zn(II) Metal Ion. Synthesis, Characterization and Thermal Behaviour of the Complex with Ketoprofen. Rev. Chim. 2016, 67, 38–41. [Google Scholar]
  18. De Souza, M.C.; Franco, C.H.J.; Pinheiro, C.B.; Diniz, R. Conformational Polymorphism of a Zinc Complex with Enalapril Antihypertensive Drug. Polyhedron 2014, 81, 290–297. [Google Scholar] [CrossRef]
  19. Mukherjee, G.; Ghosh, T. Metal Ion Interaction with Penicillins— Part VII: Mixed-Ligand Complex Formation of Cobalt(II), Nickel(II), Copper(II), and Zinc(II) with Amplicillin and Nucleic Bases. J. Inorg. Biochem. 1995, 59, 827–833. [Google Scholar] [CrossRef]
  20. Boughougal, A.; Cherchali, F.Z.; Messai, A.; Attik, N.; Decoret, D.; Hologne, M.; Sanglar, C.; Pilet, G.; Tommasino, J.B.; Luneau, D. New Model of Metalloantibiotic: Synthesis, Structure and Biological Activity of a Zinc(II) Mononuclear Complex Carrying Two Enrofloxacin and Sulfadiazine Antibiotics. New J. Chem. 2018, 42, 15346–15352. [Google Scholar] [CrossRef]
  21. Biswas, B.; Kole, N.; Patra, M.; Dutta, S.; Ganguly, M. Synthesis, Structural Characterization and Biological Activity of a Trinuclear Zinc(II) Complex: DNA Interaction Study and Antimicrobial Activity. J. Chem. Sci. 2013, 125, 1445–1453. [Google Scholar] [CrossRef] [Green Version]
  22. Margosis, M.; Lambert, J.P.F. Determination of Zinc in Pharmaceutical Products by Neutron Activation Analysis. J. Pharm. Sci. 1971, 60, 592–594. [Google Scholar] [CrossRef] [PubMed]
  23. Moody, R.R.; Taylor, R.B. Determination of Zinc in Pharmaceutical Preparations by Atomic Absorption Spectrophotometry. J. Pharm. Pharmacol. 1972, 24, 848–852. [Google Scholar] [CrossRef] [PubMed]
  24. Sweileh, J.A. On-Line Flow Injection Solid Sample Introduction Digestion and Analysis: Spectrophotometric and Atomic Absorption Determination of Iron, Copper and Zinc in Multi-Vitamin Tablets. Microchem. J. 2000, 65, 87–95. [Google Scholar] [CrossRef]
  25. Lutka, A.; Maruszewska, M. Voltammetric Determination of Copper in Selected Pharmaceutical Preparations—Validation of the Method. Acta Pol. Pharm.-Drug Res. 2011, 68, 179–184. [Google Scholar]
  26. Szlósarczyk, M.; Nowakowska, I.; Piech, R.; Opoka, W.; Krzek, J. Voltammetric Determination of Trace Elements (Cu, Pb, Zn) in Peloid-Based Pharmaceuticals. Acta Pol. Pharm. Drug Res. 2017, 74, 1373–1377. [Google Scholar]
  27. Dubey, R.K.; Puri, B.K. Differential-Pulse Polarographic Determination of Zinc and Manganese in Various Pharmaceutical and Biological Samples after Adsorption of Their Morpholine-4-Carbodithioates on Microcrystalline Naphthalene. Analyst 1994, 119, 1567–1570. [Google Scholar] [CrossRef]
  28. Matíes, R.; Jiménez, F.; Arias, J.J.; Román, M. Spectrofluorimetric Determination of Zinc with 1,2,4-Trihydroxyanthraquinone in Pharmaceutical Preparations. Anal. Lett. 1997, 30, 2059–2070. [Google Scholar] [CrossRef]
  29. Van Staden, J.F.; Hattingh, C.J. Determination of Zinc in Pharmaceutical Products Using an Electrodialyser Incorporated into a Flow System. Fresenius’ J. Anal. Chem. 2000, 367, 79–83. [Google Scholar] [CrossRef]
  30. Mohamed, S.K. Ion-Selective Electrode for Zinc Determination in Pharmaceutical Analysis. Anal. Chem. 2009, 8, 350–354. [Google Scholar]
  31. Ribeiro, M.M.A.C.; Prado, A.A.; Domingues Batista, A.; Alejandro Abarza Munoz, R.; Mathias Richter, E. Rapid Method for Simultaneous Determination of Ascorbic Acid and Zinc in Effervescent Tablets by Capillary Zone Electrophoresis with Contactless Conductivity Detection. J. Sep. Sci. 2019, 42, 754–759. [Google Scholar] [CrossRef] [PubMed]
  32. Teixeira, L.S.G.; Rocha, F.R.P.; Korn, M.; Reis, B.F.; Ferreira, S.L.C.; Costa, A.C.S. Flow-Injection Solid-Phase Spectrophotometry for the Determination of Zinc in Pharmaceutical Preparations. Anal. Chim. Acta 1999, 383, 309–315. [Google Scholar] [CrossRef]
  33. Dias, T.R.; Reis, B.F. A LED Based Photometer for Solid Phase Photometry: Zinc Determination in Pharmaceutical Preparation Employing a Multicommuted Flow Analysis Approach. J. Braz. Chem. Soc. 2012, 23, 1515–1522. [Google Scholar] [CrossRef] [Green Version]
  34. Sivaramaiah, S.; Reddy, P.R. Direct and Derivative Spectrophotometric Determination of Zinc with 2,4-Dihydroxybenzaldehyde Isonicotinoyl Hydrazone in Potable Water and Pharmaceutical Samples. J. Anal. Chem. 2005, 60, 828–832. [Google Scholar] [CrossRef]
  35. Karpińska, J.; Kulikowska, M. Simultaneous Determination of Zinc(II), Manganese(II) and Iron(II) in Pharmaceutical Preparations. J. Pharm. Biomed. Anal. 2002, 29, 153–158. [Google Scholar] [CrossRef]
  36. Bhalotra, A.; Puri, B.K. Trace Determination of Zinc in Standard Alloys, Environmental and Pharmaceutical Samples by Fourth Derivative Spectrophotometry Using 1-2-(Thiazolylazo)-2-Naphthol as Reagent and Ammonium Tetraphenylborate Supported on Naphthalene as Adsorbent. Talanta 1999, 49, 485–493. [Google Scholar] [CrossRef]
  37. Vieira, L.E.M.; Vieira, F.P.; Ávila-Terra, L.H.S.; Gaubeur, I.; Guekezian, M.; Suárez-Iha, M.E.V. Spectrophotometric Determination of Zinc in Pharmaceutical and Biological Samples with Di-2-pyridyl Ketone Benzoylhydrazone: A Simple, Fast, Accurate and Sensitive Method. Anal. Lett. 2008, 41, 779–788. [Google Scholar] [CrossRef]
  38. Zareba, S.; Pomykalski, A.; Marzec, Z. Spectrophotometric Determination of Fe(II) and Zn(II) in the Pharmaceutical Multivitamin Preparations with Microelements. Acta Pol. Pharm. 2004, 61, 175–179. [Google Scholar]
  39. Malvankar, P.L.; Shinde, V.M. Ion-Pair Extraction and Determination of Copper(II) and Zinc(II) in Environmental and Pharmaceutical Samples. Analyst 1991, 116, 1081–1084. [Google Scholar] [CrossRef]
  40. Korn, M.D.G.A.; Ferreira, A.C.; Teixeira, L.S.G.; Costa, A.C.S. Spectrophotometric Determination of Zinc Using 7-(4-Nitrophenylazo)-8-Hydroxyquinoline-5-Sulfonic Acid. J. Braz. Chem. Soc. 1999, 10, 46–50. [Google Scholar] [CrossRef] [Green Version]
  41. Islam, M.T.; Ahmed, M.J. A Simple Spectrophotometric Method for the Trace Determination of Zinc in Some Real, Environmental, Biological, Pharmaceutical, Milk and Soil Samples Using 5,7- Dibromo-8-Hydroxyquinoline. Pakistan J. Anal. Environ. Chem. 2013, 14, 1–15. [Google Scholar]
  42. Gaubeur, I.; da CunhaAreias, M.C.; Avila Terra, L.H.S.; Suárez-Iha, M.E.V. Spectrophotometric Determination of Zinc in Pharmaceutical Samples Using Di-2-Pyridyl Ketone Salicyloylhydrazone. Spectrosc. Lett. 2002, 35, 455–465. [Google Scholar] [CrossRef]
  43. Gaubeur, I.; Ávila-Terra, L.H.S.; Masini, J.C.; Suárez-Iha, M.E.V. Spectrophotometric Flow Injection Methods for Zinc Determination in Pharmaceutical and Biological Samples. Anal. Sci. 2007, 23, 1227–1231. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Capitán, F.; Cuenca, M.J.; Espinosa, P.; Capitán Vallvey, L.F. Spectrophotometric Determination of Zinc with 2-(2-Thienyl) Benzothiazoline in Pharmaceutical Samples. Bull. Sociétés Chim. Belges 1988, 97, 235–236. [Google Scholar] [CrossRef]
  45. Asuero, A.G.; Rosales, D.; Rodriguez, M.M. Spectrophotometric Determination of Zinc in Pharmaceutical Preparations with Biacetyl Mono(2-Pyridyl)-Hydrazone. Microchem. J. 1984, 30, 33–38. [Google Scholar] [CrossRef]
  46. Amin, A.S. Spectrophotometric Determination of Zinc in Pharmaceutical Samples with Some Salicylic Azo Compounds. Anal. Lett. 1994, 27, 95–102. [Google Scholar] [CrossRef]
  47. Zarȩba, S. Spectrophotometric Determination of Zinc in Pharmaceutical Multimineral Preparations by Azo-Dye Derivatives of Benzimidazole and 1,2,4-Triazole. Pharm. Acta Helv. 1995, 70, 195–198. [Google Scholar] [CrossRef]
  48. Themelis, D.G.; Tzanavaras, P.D.; Liakou, A.A.; Tzanavaras, H.D.; Papadimitriou, J.K. Normal and Differential Demasking Flow-Injection Manifold for the Direct Spectrophotometric Determination of Zinc(II) in Biological Materials and Pharmaceutical Formulations. Analyst 2000, 125, 2106–2111. [Google Scholar] [CrossRef]
  49. Van Staden, J.K.F.; Tsanwani, M. Determination of Zinc in Pharmaceutical Products by Use of a Sequential Injection Analysis System. Fresenius’ J. Anal. Chem. 2001, 371, 376–379. [Google Scholar] [CrossRef]
  50. Nagarjunareddy, D.; Reddy, K.V.; Reddy, K.H. Simple and Sensitive Spectrophotometric Determination of Zn (II) in Biological and Pharmaceutical Samples with 2-Benzoylpyridine Thiosemicarbazone(BPT). J. Chem. Pharm. Res 2011, 3, 205–213. [Google Scholar]
  51. Raman, B.; Shinde, V.M. Extraction and Determination of Zinc in Pharmaceutical Samples. Talanta 1989, 36, 469–472. [Google Scholar] [CrossRef]
  52. Najim, S.S.; Abid, M.; Hameed, A.-H.; Al-Shakban, A.; Fandi, T.S. Spectrophotometric Determination of Zinc in Pharmaceutical Medication Samples Using 8-Hydroxyquinoline Reagent. Int. J. Chem. 2020, 12, 29–36. [Google Scholar] [CrossRef] [Green Version]
  53. Muginova, S.V.; Zhavoronkova, A.M.; Polyakov, A.E.; Shekhovtsova, T.N. Application of Alkaline Phosphatases from Different Sources in Pharmaceutical and Clinical Analysis for the Determination of Their Cofactors; Zinc and Magnesium Ions. Anal. Sci. 2007, 23, 357–363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Mohammed, A.F.; Baqir, S.J. Spectrophotometric Determination of Zinc in Pharmaceutical Preparations by Using a New Synthesized Reagent [4,5-Diphenyl-2-((1E)-(4-(1-(2-Phenylhydrazono)Ethyl)Phenyl)Diazenyl)-4H-Imidazole]. Iraqi Natl. J. Chem. 2015, 15, 271–283. [Google Scholar]
  55. United States Pharmacopeial Convention (USP). United States Pharmacopeia and National Formulary (USP 41-NF 36); United States Pharmacopeial Convention: Rockville, MD, USA, 2016. [Google Scholar]
  56. Vani, D.S.; Subudhi, K.S.; Shivaramaiah, S.; Rao, K.A. Direct and Derivative Spectrophotometric Determination of Zn(II) in Pharmaceuticals. Chem. Sci. Trans. 2013, 2, 513–523. [Google Scholar] [CrossRef]
  57. Benamor, M.; Belhamel, K.; Draa, M.T. Use of Xylenol Orange and Cetylpyridinium Chloride in Rapid Spectrophotometric Determination of Zinc in Pharmaceutical Products. J. Pharm. Biomed. Anal. 2000, 23, 1033–1038. [Google Scholar] [CrossRef]
  58. Heghes, S.C.; Rus, L.M.; Rus, L.L.; Bojita, M.T.; Iuga, C.A. HPLC-UV Determination of Indapamide in the Presence of Its Main Synthesis and Degradation Impurities. Method Validation. Farmacia 2017, 65, 5. [Google Scholar]
  59. Gligor, F.G.; Frum, A.; Vicaș, L.G.; Totan, M.; Roman-Filip, C.; Dobrea, C.M. Determination of a Mixture of Plantago Lanceolata L. and Salvia Officinalis L. by High-Performance Liquid Chromatography with Ultraviolet Detection (HPLC-UV). Anal. Lett. 2020, 53, 1391–1406. [Google Scholar] [CrossRef]
  60. Avram, N.; Heghes, S.C.; Rus, L.L.; Juncan, A.M.; Rus, L.M.; Filip, L.; Filip, C.R. HPLC-UV Determination of Dextromethorphan in Syrup Method Validation. Rev. Chim. 2019, 70, 487–490. [Google Scholar] [CrossRef]
  61. ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-2-r1-validation-analytical-procedures-text-methodology-step-5_en.pdf (accessed on 2 February 2022).
  62. Pinto, J.J.; García-Vargas, M.; Moreno, C. Enhanced Spectrophotometric Methods for Trace Metal Determination in Waters: Zinc as an Example. Anal. Methods 2012, 4, 147–152. [Google Scholar] [CrossRef]
  63. Mehta, P.S.; Patel, V.B. Spectrophotometric Method for Determination of Fe(II) and Zn(II) in Multivitamin Soft Gel Capsule. Int. J. Pharm. Res. Anal. 2012, 2, 87–91. [Google Scholar]
Applsci 12 04476 g001 550
Figure 1. Overlaid spectra for calibration solutions, LOQ, and tablet sample.
Figure 1. Overlaid spectra for calibration solutions, LOQ, and tablet sample.
Applsci 12 04476 g001
Table
Table 1. Statistical evaluation of linearity.
Table 1. Statistical evaluation of linearity.
ParameterValues for EtalonValues for SMDSCTheoretical ValuesAcceptance Criteria
Correlation coefficient (r)0.99990.9998--
Intercept0.000090.0007--
Slope0.382120.38094--
Intragroup variance homogeneity (Cochrane test)0.57450.44Ctheor = 0.68C < Ctheor
Significant slope (Fisher test)420,144.12307,623.37Ftheor = 4.67F > Ftheor
Regression curve validity (Fisher test)0.280.19Ftheor = 3.71F < Ftheor
Intercept comparison with null (t-Student test)−0.1406−0.895ttheor = 2.16t < ttheor
Regression curves slopes comparison (t-Student test)-1.2998ttheor = 2.056t < ttheor
Intercepts comparison (t-Student test)-1.1747ttheor = 2.056t < ttheor
Table
Table 2. Statistical evaluation of precision.
Table 2. Statistical evaluation of precision.
ParameterCalculated ValuesTheoretical ValuesAcceptance Criteria
Intragroup variance homogeneity (Cochrane test)C = 0.562Ctheor = 0.68C < Ctheor
Repeatability variation coefficient (CVr%)1.57%
Reproducibility variation coefficient (CVR%)1.77%
Table
Table 3. Statistical evaluation of accuracy.
Table 3. Statistical evaluation of accuracy.
ParameterCalculated ValuesTheoretical ValuesAcceptance Criteria
Intragroup variance homogeneity (Cochrane test)C = 0.622Ctheor = 0.68C < Ctheor
Medium recovery validity (Fischer test)F = 1.653Ftheor = 3.48F < Ftheor
Confidence interval of the mean recovery (%)98.29–100.55%
Table
Table 4. Evaluation of robustness.
Table 4. Evaluation of robustness.
Apparatus
Calibration curve
SlopeInterceptCorrelation coefficient
s10.382940.00070.9998
s20.382560.00080.9997
Zinc to other components ratio
Confidence interval of the mean recovery
Level 80%97.84–101.03%
Level 100%98.29–100.55%
Level 120%98.53–101.47%
Table
Table 5. Statistical evaluation of linearity.
Table 5. Statistical evaluation of linearity.
Industrial Batch nAAS Method Results (mg/Tablet)Proposed Method Results (mg/Tablet)Individual Differences between ResultsMean Difference between Results Standard Deviation of Differences between ResultsCalculated tTheoretical tAcceptance Criteria
15.45.380.020.02750.041132.1143.18t < ttheor
24.84.770.03
354.960.08
44.94.82−0.02
Table
Table 6. Current analytical method comparison with other published spectrophotometric methods.
Table 6. Current analytical method comparison with other published spectrophotometric methods.
Complexing AgentRangeCorrelation CoefficientWavelengthSample PreparationLODReference
Di-2-pyridyl Ketone Benzoylhydrazone0.020–1.82 µg/mL0.9994320 nmDigestion in concentrated HCl/no L/L extraction involved2.29 mg/L[37]
3-mercapto-5-(2,4-dihydroxy-3-carboxyphenylazo-1)-1,2,4-triazole0.05–2.7 µg/mL0.9999480 nmDigestion in concentrated H2SO4 and H2O2/no L/L extraction involved0.04 mg/L[38]
Dithizone2–12 µg/mL0.9999525 nmDissolution in 0.1 N HCl/L/L extraction in chloroform involved111 mg/L[63]
2-(2′,4′-dihydroxyphenylazo-1′) benzimidazole0.06–1.44 µg/mL0.9998540 nmDissolution in 0.1 N HCl/no L/L extraction involvedNR[47]
3-mercapto-5-(2′-hydroxynaphtylazo-1′)-1,2,4-triazole0.06–2.65 µg/mL0.9994530 nmDissolution in 0.1 N HCl/no L/L extraction involvedNR[47]
3-(5′-mercapto-1′,2′,4′-triazole-3′-azo)-2,4-dihydroxybenzoic acid0.06–2.7 µg/mL0.9999480 nmDissolution in 0.1 N HCl/no L/L extraction involvedNR[47]
2,2A-dipyridyl-2-pyridylhydrazone *0–10 µg/mLNR448 nmDissolution in 0.1 N HCl/no L/L extraction involved6 µg/L[48]
Xylenol orange *10–60 µg/mL0.9984568 nmDissolution in deionized water/no L/L extraction involved420 µg/L[49]
2-Benzoylpyridine thiosemicarbazone0.26–2.61 µg/mLNR430 nmDigestion in concentrated HNO3/no L/L extraction involved64 µg/L[50]
1-(2-pyridylazo)-2-naphthol0.5–4 µg/mLNR555 nmDigestion in aqua regia/L/L extraction in toluene NR[51]
8-Hydroxy quinoline1–5 µg/mL0.9979384 nmDigestion in concentrated HNO3/no L/L extraction involved0.381 mg/L[52]
4,5-diphenyl-2-((1E)-(4-(1-(2-phenylhydrazono)ethyl)phenyl)diazenyl)-4H-imidazole0.5–27 µg/mL0.9997520 nmDissolution in 2 N HCl/no L/L extraction involved0.12 mg/L[54]
1 -(Z-pyridylazo)-Z-naphtholNRNR560 nmDigestion in concentrated HCl/L/L extraction in toluene involvedNR[39]
7-(4-nitrophenylazo)-8-hydroxyquinoline-5-sulfonic acid0.05–1.0 µg/mL0.9996520 nmCalcination and digestion in concentrated HCl/no L/L extraction involved0.015 mg/L[40]
5,7-dibromo-8-hydroxyquinoline0.02–4 µg/mL0.9998391 nmDigestion with concentrated HNO3/no L/L extraction involved5 µg/L[41]
di-2-pyridyl ketone salicyloylhydrazone0.01308–1.308 µg/mLNR376 nmDigestion in concentrated H2SO4 and H2O2/no L/L extraction involved4.06 µg/L[42]
di-2-pyridyl ketone salicyloylhydrazone *0.217–4.6 µg/mL−1NR376 nmDigestion in concentrated HCl/L/L extraction in toluene involved13.9 µg/L[43]
2-(2-thienyl) benzothiazoline0.2–7 µg/mLNR465 nmDigestion in concentrated H2SO4/L/L extraction in chloroform involved40 µg/L[44]
Xylenol orange0.5–1.5 µg/mL0.9998560 nmDissolution in 0.1 N HCl/no L/L extraction involved4.93 µg/LPresented method
NR—not reported. * Flow-injection analytical method; L/L—liquid–liquid.
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Rus, L.L.; Juncan, A.M.; Crăciun, V.I.; Frum, A.; Hegheș, S.-C.; Butuca, A.; Dobrea, C.M.; Chis, A.A.; Muntean, A.C.; Vonica-Ţincu, A.L.; et al. Determination of Zn2+ in Solid Pharmaceutical Dosage Forms by Means of Spectrophotometry in Micellar Media: Method Validation. Appl. Sci. 2022, 12, 4476. https://doi.org/10.3390/app12094476

AMA Style

Rus LL, Juncan AM, Crăciun VI, Frum A, Hegheș S-C, Butuca A, Dobrea CM, Chis AA, Muntean AC, Vonica-Ţincu AL, et al. Determination of Zn2+ in Solid Pharmaceutical Dosage Forms by Means of Spectrophotometry in Micellar Media: Method Validation. Applied Sciences. 2022; 12(9):4476. https://doi.org/10.3390/app12094476

Chicago/Turabian Style

Rus, Luca Liviu, Anca Maria Juncan, Veronica Isabela Crăciun, Adina Frum, Simona-Codruța Hegheș, Anca Butuca, Carmen Maximiliana Dobrea, Adriana Aurelia Chis, Andrei Catalin Muntean, Andreea Loredana Vonica-Ţincu, and et al. 2022. "Determination of Zn2+ in Solid Pharmaceutical Dosage Forms by Means of Spectrophotometry in Micellar Media: Method Validation" Applied Sciences 12, no. 9: 4476. https://doi.org/10.3390/app12094476

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