Comparative study of several Analytical methods for determination of Manganese content in some dietary supplements in Syrian market

 

Maya Tawfik Razzouk¹*, Fidaa Am Ali²

¹Master Student, Department of Analytical and Food Chemistry, Faculty of Pharmacy,

University of Damascus, Syrian Arab Republic.

²Prof. A. Dept. of Analytical and Food Chemistry, Faculty of Pharmacy,

University of Damascus, Syrian Arab Republic.

 

ABSTRACT:

In this study, three different analytical methods for manganese determination (volumetric, spectrophotometric, FAAS) were compared regarding sensitivity, accuracy, specificity and cost. The spectrophotometric method was chosen to analyze pharmaceutical formulations which are available in Syrian markets as Dietary supplements containing many minerals including manganese. Manganese was determined first quantitatively by complexometric direct titration with EDTA at pH 10 using Eriochrome black T as an indicator, the linearity range was 16-26µg/ml (R²=0.9963). The precision as RSD was <2%, and accuracy was determined as recovery which ranged between 100.18 – 105.31%. The spectrophotometric method depends on manganese oxidation to permanganate. The absorbance of the resulting permanganate solution is measured by visible-spectrophotometer at the maximum absorption wavelength (λ max) at 525nm. Beer’s law is obeyed over the concentration ranges of 5-30µg/ml (R²=0.9971). The precision as RSD was <2%, and accuracy was determined as recovery which ranged between 93.94 – 103.13%. Previous methods were compared to one of the most sensitive but costly processes to measure manganese by Atomic Absorption Spectroscopy (AAS) using a manganese bulb with the maximum absorption wavelength (λ max) at 279.5nm. Beer’s law is obeyed over the concentration ranges of (0.5-3μg/ml) and the correlation coefficient value (R2 = 0.9993). The precision as RSD was <2% and accuracy was determined as recovery which ranged between 97.24 and 100.04%. Manganese content in a number of dietary supplements samples was estimated, and the results did not coincide with the addressed limits on the labelled cards and the authorized global values.

 

 

 

INTRODUCTION:

The use of Dietary supplements in general and multivitamins-multiminerals in particular is prevalent and growing randomly in Syria and wild world in the last few years. Although multivitamins, multiminerals (MVM) and similar terms (eg, multis or multiples) are commonly used, they have no standard scientific, regulatory, or marketplace definitions. MVM composition databases use label values as surrogates for analyzed values. However, actual vitamin and mineral amounts often deviate from label values¹.

 

FDA reports defines Dietary supplements as orally taken products that contain a "dietary ingredient including vitamins, minerals, amino acids, and herbs or botanicals, as well as other substances that can be used to supplement the diet²., meanwhile the Agency for Healthcare Research and Quality in USA defined multivitamin/mineral supplements as any supplements containing 3 or more vitamins and/or minerals without herbs, hormones, or drugs, each at a dose less than the UL determined by the Food and Nutrition Board³.

 

Manganese is a nutritionally essential mineral but it is potentially toxic⁴. It is involved in many physiological processes in the body. It is located largely in the mitochondria⁵ and plays an important role in a number of physiologic processes as a constituent of multiple enzymes and an activator of other enzymes⁶. Also, it is involved in neurological function^7,8. and essentially required for normal thyroid function and thyroxin formation⁹.

 

Because of the rising consumption of this kind of products in Syria and the high cost of most modern devices that provide the selectivity required for the element to be analyzed in this type of preparations. The objective of this study was to compare between three different analytical methods for manganese determination (spectrophotometric, volumetric, AAS) to study the possibility of analyzing it in alternative methods, As well as comparing the analysis results of several samples with the labelled values, especially that Dietary supplements do not undergo drug control in many countries around the world.

 

MATERIALS AND METHODS:

INSTRUMENTATION:

UV/VIS Spectrophotometer (single beam – Scinco S-3100)

Atomic Absorption Spectroscopy (AAS) (ZEE nit7 700P)

Manganese hollow cathode lamp

Magnetic stirrer

A SHIMADZU analytical balance with sensitivity 0.01 mg.

 

Chemicals and Reagents:

Manganese ion Standard Solution 1000ppm (Merck), Distilled water, Eriochrome Black (HIMEDIA), Ascorbic Acid (Shenzhen China CO- LTD), Disodium Ethylene Diamine Tetra Acetic Acid (EDTA-Na) (Sigma Aldrich), Deionized water, Phosphoric Acid 85% (Merck), Potassium Periodate (M&B Laboratory Chemicals), Nitric Acid 65% (Panereac), Ammonia (Panereac), Ammonium chloride (Merck), Dietary supplements samples from Syrian markets for three local companies A, B, C

 

Company A: Each tablet contains: vitamin D3 200 IU, Calcium (as carbonate) 600mg, magnesium (as oxide) 40mg, copper (as sulfate) 1mg, zinc (as oxide) 7.5mg, manganese (as sulfate) 1.8mg, Lot: 2620, 2627

 

Company B: Each tablet contains: vitamin D3 200 IU, Calcium (as carbonate) 600mg, magnesium (as oxide) 40mg, copper (as sulfate) 1mg, zinc (as oxide) 7.5mg, manganese (as sulfate) 1.8mg, Boron (as sodium borate) 250mcg, Lot: 6087، 6088، 6089

 

Company C: Each tablet contains: vitamin D3 200 IU, Calcium (as carbonate) 600mg, magnesium (as oxide) 40mg, copper (as sulfate) 1mg, zinc (as oxide) 7.5mg, manganese (as sulfate) 1.8mg, Boron (as sodium borate) 250mcg, Lot: 869، 806، 867

·       Preparation of chemicals for Volumetric method:¹⁰

Preparation of Buffer Solution (Ammonia- Ammonium Chloride) pH=10:

67.5g of Ammonium Chloride were dissolved in 57ml of Ammonia in a 1000ml volumetric flask and the volume was diluted with distilled water.

 

Preparation of 0.01M EDTA:

3.72g of EDTA were dissolved in 900ml of distilled water in 1000ml volumetric flask and the volume was diluted with distilled water.

 

Preparation of 0.05M Zinc Ion Solution for EDTA Standardization:

0.68g of zinc chloride were dissolved with distilled water in 100ml volumetric flask.

 

Standardization of an EDTA Solution with Zinc Ion Solution:

5ml of zinc ion solution were put in a beaker and 1ml of buffer solution (ammonia-ammonium chloride) pH=10 was added with few drops of Eriochrome Black T, the mix was titrated with pre-prepared EDTA

 

Preparation of Eriochrome Black T:

0.5g of Eriochrome Black and 4.5g hydroxylamine hydrochloride were dissolved in 90ml ethanol 95% in 100ml volumetric flask and the volume was diluted with distilled water.

 

·       Procedure for volumetric method:

A suitable volume (1.6, 1.8, 2, 2.2, 2.4, 2.6ml) of manganese ion standard solution (1000ppm) were transferred in a series of 100ml volumetric flask and diluted with distilled water to the volume.

 

Each flask content was titrated with 0.1M Na₂EDTA after adding 1mg Ascorbic Acid and 10ml of Buffer Solution (Ammonia- Ammonium Chloride) with few drops of Eriochrome Black T as an indicator.

 

·       Preparation of chemicals for spectrophotometric method:^11,12

Preparation of phosphoric acid 25% :

30ml of 85% commercial phosphoric acid were transferred in a 100ml volumetric flask and diluted with distilled water to the volume.

 

Preparation of 500ppm manganese-working solution:

25ml of manganese standard solution were transferred in a 50ml volumetric flask and diluted with distilled water to the volume.

 

·       Procedure for spectrophotometric method:

A suitable volumes (1, 2, 3, 4, 5, 6ml) of manganese -working solution (500ppm) were transferred in a series of 100ml volumetric flask, 10 ml of distilled water and 10ml of phosphoric acid 25% were added with 20cg of potassium periodate.

 

The flasks content were boiled for 2 minutes and were cooled to room temperature, then each flask was diluted with distilled water to the volume.

 

The absorbance of resulting colored solutions was measured by visible-spectrophotometer at the maximum absorption wavelength λ max = 525nm.

 

·       Preparation of chemicals for FAAS method:¹³

Preparation of nitric acid / water solution 1%:

1.5ml of nitric acid were transferred in a 100ml volumetric flask and diluted with de-electrolyte water (drip distillation) to the volume.

 

Preparation of 10 ppm Manganese working solution:

0.5ml of manganese standard solution were transferred in a 50ml volumetric flask and the volume was supplemented with nitric acid / water solution 1%.

 

·       Procedure for FAAS method:

A suitable volume (2.5, 5, 7.5, 10, 12.5, 15ml) of manganese -working solution (10ppm) were transferred in a series of 50ml volumetric flask and diluted with distilled water to the volume. Then they were measured by Atomic Absorption Spectroscopy (AAS) using a manganese bulb according to the conditions in table 1.

 

Table 1: work conditions on FAAS

 

·       Procedure for commercial tablets:

Ten tablets of labeled claim 2mg of manganese were weighed precisely. An average weight of each tablet was determined. An accurately weighed quantity of powder equivalent to 2mg of manganese was dissolved with 10ml distilled water and 10ml phosphoric acid 25%. The samples were mixed well with Ultrasonic then filtered and 20cg of potassium periodate were added to be boiled for 2 minutes. The samples were cooled and diluted with distilled water to the volume.

 

The absorbance was measured with visible spectrophotometer λ max = 525nm and manganese concentration was calculated using calibration curve.

 

 

RESULTS AND DISCUSSION:

Absorption spectra for Permnganate in Spectrophotometer:

Permnganate (which resulted from manganese oxidation with potassium periodate and phosphoric acid 25%) showed maximum absorption at 525nm.

 

Validation of the methods¹⁴:

Linearity:

In the volumetric method, the correlation coefficient (R²) was 0.9963 over the concentration ranges of 16-26µg/ml and in spectrophotometric method Beer’s law was obeyed over the concentration ranges of 5-30µg/ml (R2=0.9971) while in AAS, linearity was shown between 0.5-3µg/ml (R²=0.9993)

 

The three analytical methods indicated good linearity (Figures 2,3,4). The limit of detection (LOD) and limit of Quantification (LOQ) were calculated using the following equations:

 

LOD = 3.3 σ / S, LOQ = 10 σ / S

 

Where σ is the standard deviation of intercept. S is the slope of calibration curve. The results are summarized in table1.

 

 

Figure1: Calibration graph for Manganese determination in volumetric method

 

 

Figure 3: Calibration graph for Manganese determination in AAS

 

Figure 2: Calibration graph for Manganese determination in Spectrophotometric method

 

Table 2: Quantitative parameters of the three analytical methods for Manganese determination

*Y = aX+ b, where X is the concentration of Manganese μg ml^-1

 

Table 3: Accuracy Comparison for the three analytical methods for Manganese determination

*n=3

 

Accuracy and Precision:

In the three analytical methods, Accuracy was determined for three concentration levels, three replicates and the results were recorded as percent recovery.

 

Precision was estimated at one concentration level, 6 replicates and the results were recorded as relative standard deviation. The calculated relative standard deviations were below 2% indicating good precision of the results are summarized in table 3, 4.

 

Table 4: Precision comparison for the three analytical methods for Manganese determination

*RSD=Relative standard deviation

 

Specificity and effect of excipients:

The specificity of the methods was investigated by observing any interferences encountered from the common tablet excipients, such as Talc, sodium loryl sulfate, methyl cellulose, starch, magnesium stearate. These excipients did not interfere with the compared methods.

 

Cations presence effect on the spectrophotometric method:

To make sure there is no overlap and to avoid any interference with other associated cations in the pharmaceutical formulations, the spectrophotometric method was applied on standard concentrations of manganese-associated minerals within the pharmaceutical forms (zinc, magnesium, calcium and copper) (each cation standard separately) and mixtures from (cation standard -manganese standard) and no color was shown. The change of manganese concentration was negligible indicating the selectivity of the analytical method.

 

Assay of tablets:

Samples were analyzed by visible spectrophotometer, the manganese content of company A, B, C tablets was 95.61%, 53.62%, 2.012 % respectively of the label claim, and there were no significant differences when the previous samples were analyzed by the atomic absorption spectroscopy. The results are summarized in Table (5).

 

 

Table 5: Analyzed samples results with two methods (Colorimetric, FAAS)

Company	Labeled amount (mg/tab)	Amount obtained by FAAS method	Percentage recovery*	Amount obtained by spectrophotometric method	Percentage recovery*
A	1.8	1.74	97.14	1.72	95.61
B		0.97	54.06	0.96	53.62
C		0.036	2.04	0.036	2.012

n=24 (8 for each company)

 

CONCLUSION:

The validity of the three analytical methods was verified for manganese determination. However, the spectrophotometric method was favorable to estimate manganese in dietary supplements tablets because of the wide range of cation-manganese interactions in the complexometric titration which require multi-step titration using masking agents to mask other cations that could interact with manganese determination. The determination of manganese in commercial tablets was performed by spectrophotometer comparing with atomic absorption spectroscopy. The results showed no significant differences between the two methods even in the presence of other cations in commercial formulations, and therefore the atomic absorption spectroscopy method could be replaced by the spectrophotometric method using visible spectrophotometer, which has wide availability in quality control laboratories and could be a valid method for routine manganese determination.

 

Stricter quality control is required over Dietary supplements in the Syrian market to ensure their compliance with label claims.

 

REFERENCES:

1.      Yetley EA. Multivitamin and multimineral dietary supplements: definitions, characterization, bioavailability, and drug interactions. The American Journal of Clinical Nutrition. 2007; 85(1):269S-276S.

2.      (FDA Reports: FDA 101: Dietary Supplements) Available from URL: https://www.fda.gov/consumers/consumer-updates/fda-101-dietary-supplements

3.      Huang H-Y et al. Multivitamin/Mineral Supplements and Prevention of Chronic Disease: Executive Summary. The American Journal of Clinical Nutrition.2007; 85(1), 265S–268S

4.      Keen CL et al. Nutritional aspects of manganese from experimental studies. Neurotoxicology. 1999; 20(2-3):213-223.

5.      Watts D.L. The Nutritional Relationships of Manganese. Journal of Orthomolecular Medicine. 1990; 5(4),219-222

6.      Nielsen FH. Ultratrace minerals. Modern Nutrition in Health and Disease, edited by Shils M, Olson JA, Shike M, Ross AC. Williams & Wilkins, Baltimore. 1999; 9th ed. pp283-303.

7.      Wedler FC. Biochemical and nutritional role of manganese: an overview. Manganese in health and disease. edited by Klimis-Tavantzis DJ. CRC Press, Boca Raton. 1199.pp1-37.

8.      Albrecht J et al. Glutamine in the central nervous system: function and dysfunction. Front Biosci. 2007; 12:332-343.

9.      Soldin OP and Aschner M. Effects of manganese on thyroid hormone homeostasis. Neuro Toxicology, 2007; 28(5), 951-956

10.   USP 41-NF 36, Monograph, manganese sulfate

11.   Mehlig, J. P. Colorimetric determination of manganese with periodate. Industrial & Engineering Chemistry Analytical Edition.1939; 11(5), 274–277.

12.   Willard, H. H and Greathouse, L. H. The Colorimetric Determination of Manganese by Oxidation with Periodate. Journal of the American Chemical Society, 1917; 39(11), 2366–2377.

13.   Analytical Methods for Atomic Absorption Spectroscopy, The Perkin-Elmer Corporation 1996,4-10

14.   ICH harmonised tripartite guideline, validation of analytical procedures: text and methodology, Q2R1, International Conference on Harmonization of Technical requirements for Registration of Pharmaceuticals for Human use, 2005.

 

 

 

Received on 19.12.2019           Modified on 24.02.2020

Accepted on 21.04.2020         © RJPT All right reserved