1. INTRODUCTION:

Zinc and Manganese are essential trace elements frequently included in multivitamin and multimineral syrups due to their vital roles in various biological and physiological processes¹. These elements are necessary for numerous enzyme systems and metabolic functions, making them critical for maintaining human health². In the United States, it is estimated that approximately 40% of the population regularly uses dietary supplements containing vitamins and minerals, highlighting the importance of ensuring the safety and quality of such products³. Manganese contributes significantly to normal development and is involved in the metabolism of carbohydrates, lipids and proteins⁴. Zinc, on the other hand, has been shown to reduce oxidative stress and inflammatory markers, and its supplementation has been associated with a lower risk of certain age-related conditions, including pneumonia and macular degeneration^5,6. Since the human body cannot synthesize these minerals, they must be obtained through diet or supplementation⁷. However, exceeding recommended daily intake levels can lead to adverse health effects^8,9. The tolerable upper intake levels for Manganese typically range from 2 to 5mg per day, while Zinc recommendations are 8mg per day for adult women and 11mg for adult men¹⁰. Excess Zinc intake has been linked to weakened immune response, reduced HDL cholesterol and even neurological symptoms such as seizures¹¹. Similarly, chronic exposure to elevated levels of Manganese may result in neurotoxicity and symptoms resembling Parkinson’s disease¹². Given their inclusion in multicomponent formulations and the potential health risks of excessive intake, accurate and reliable quantification of these elements in final products is essential. Although Atomic Absorption Spectrometry (AAS) is a widely used technique due to its affordability and ease of use, it often lacks the sensitivity needed to detect trace metal ions at extremely low concentrations, particularly in complex matrices like syrups¹³. A review of existing literature reveals an absence of validated methods capable of measuring both Zinc and Manganese simultaneously in combination products.

This study focuses on the development and validation of a straightforward, reproducible and precise method for the simultaneous determination of Zinc and Manganese in a multivitamin and multimineral syrup using atomic absorption spectrophotometry. The validation was performed in accordance with the International Council for Harmonisation (ICH) guidelines. Additionally, the method complies with the specifications set by the United States Pharmacopeia (USP) for oral solutions containing water-soluble vitamins and minerals, which require the content of active ingredients to fall within the range of not less than 90.0% and not more than 125.0% of the labelled claim for each mineral¹⁴.

MATERIALS AND METHODS:

Chemicals and reagents:

Bevon Suspension^® is a Multivitamin, Multimineral and Antioxidant Suspension manufactured by Zuventus Healthcare Ltd. composed as each 5mL Containing 3 mg of Zinc and 0.8mg of Manganese along with other nutrients. The placebo and formulation (sample) were made available from our own source as Zuventus Healthcare limited (Formulation development centre, Pune, India). The chemicals, standard solutions, used were procured from various sources. Nitric acid (AR grade), Hydrogen peroxide 30% (AR grade) were procured from Rankem. Hydrochloric acid (AR grade) was procured from SD Finechem. Standard solution of Zinc and Manganese was procured from Merck specialities private limited (Mumbai, India), Milli-Q grade HPLC water was used.

Instrumentation and Software’s:

An atomic absorption spectrometer of Perkin Elmer equipped Zinc hollow cathode lamp with software from Winlab 32, was set up for the flame method using acetylene and air. Weighing was done with Mettler Toledo balances and samples were heated on Hot plate made by IKA. The pH measurements were taken using a Mettler Toledo pH meter. LBS2 4.5-liter ultrasonic water bath from Falc instruments was used for heating and mixing.

Method Development Trials.

Atomic Absorption Spectrophotometer used in absorbance mode with Zinc lamp at 213.86nm and Manganese lamp at 279.48nm. Air-Acetylene flame used with 2.5L/minute flow of Acetylene; air is used as oxidant with flow of 10L/minutes. Slit for Zinc was 2.7/1.8mm and for Manganese was 1.8/0.6.

Diluent Preparation: Water used as diluent.

Standard preparation: Standard solutions of Zinc (0.2, 0.3, 0.4, 0.5, and 0.6ppm) and Manganese (0.50, 0.75, 1.00, 1.25, and 1.50ppm) were prepared by serial dilution of commercially available 1000ppm stock solutions of Zinc and Manganese, respectively.

Sample Preparation: For test solution preparation, 5 mL of the sample was subjected to acid digestion in a 500mL conical flask on a hot plate at 180–200°C using a mixture of Hydrochloric acid, Nitric acid and Hydrogen peroxide until a clear solution was obtained. The resulting digest was diluted to a final volume of 500mL with deionized water and subsequently further diluted to achieve final concentrations of 0.36ppm Zinc and 0.96 ppm Manganese for AAS analysis.

Instrumentation and Optimized Chromatographic Condition:

A robust Atomic Absorption Spectrophotometric (AAS) method was established for the determination of Zinc and Manganese in chemically digested samples. The method was optimized for sample preparation and detector sensitivity to ensure linear absorbance responses within an acceptable analytical range for both analytes.

Calibration and Linearity:

Standard stock solutions (1000ppm) of Zinc and Manganese were serially diluted to prepare working standards. The calibration ranges for Zinc was 0.2–0.6 ppm and for Manganese was 0.50–1.50ppm

Instrument sensitivity was adjusted to maintain absorbance values between 0.15 and 0.95, ensuring accurate quantification.

Instrument Configuration:

The optimized AAS operating parameters were as follows:

Zinc: Wavelength 213.9nm, slit width 1.0nm, lamp current 5mA

Manganese: Wavelength 279.5nm, slit width 0.2nm, lamp current 7mA

Flame Conditions:

Air-acetylene, burner height 7.5mm, acetylene flow rate 1.8L/minute. These conditions were selected to maximize signal intensity and stability for both elements.

Sample Digestion and Preparation Each sample (5mL) was transferred into a borosilicate digestion flask and subjected to acid digestion at 180–200°C. The digestion mixture comprised 10mL of concentrated Hydrochloric acid (HCl), 5mL of concentrated Nitric acid (HNO₃), and 2mL of 30% Hydrogen peroxide (H₂O₂). Heating was continued until the solution became transparent, indicating complete digestion.

After cooling to room temperature, the solution was diluted to 500mL with deionized water. Further dilutions were performed as necessary to bring analyte concentrations within the validated linear range. Final concentrations in the working solutions were approximately 0.36ppm for Zinc and 0.96ppm for Manganese.

Method Validation:

Analytical method validation was performed to assess specificity, linearity, precision, accuracy and solution stability following ICH guidelines Q2 (R1)¹⁵.

Specificity:

To quantitatively measure the analyte in the presence of components that may be expected to be present in the sample matrix specificity test performed for blank, placebo, standard and test solutions¹⁶. It ensures that there is no interference from diluent and/or degradation products and/or impurities and/or placebo and/or the analytes with each other¹⁷. In this investigation, specificity was evaluated by injecting Blank, Placebo, Standard and test solutions into the AAS system, followed by recording absorbance.

Linearity:

Linearity was evaluated by analyzing multiple concentrations of Zinc and Manganese standards. Zinc standard solutions were prepared at concentrations from 0.2 to 0.6ppm, while Manganese standards were prepared from 0.50 to 1.50ppm. Absorbance corresponding to each sample's concentration were recorded and concentration vs. Absorbance graph was plotted. Slope and correlation coefficient were determined.

Accuracy:

Accuracy of the AAS method was done to determine how correctly the method measures the actual concentration of an element in a sample. It was evaluated by performing recovery studies at multiple concentration levels¹⁸. (e.g., 50%, 100%, and 150%). Known amounts of the analyte were separately spiked in placebo solution in triplicate and recovery was assessed by comparing the obtained concentration with the added amount. The method met accuracy requirements, with recoveries ranging between 90% and 115%.

Precision:

System precision is determined by repeated measurements of the same parameter using the same instrument and method. It evaluates the consistency of results under identical conditions, often using standard deviation as a statistical tool. Method precision in analytical chemistry refers to the ability of a method to provide reproducible results under constant experimental settings. It reflects the reliability and repeatability of the method. Intermediate precision, also known as within-laboratory variability, examines result variation under different conditions such as different days, analysts, or instruments within the same lab. It is assessed using statistical measures like standard deviation and %RSD. High precision shows minimal variation between results, indicating good method control. Low precision indicates higher variability and may require refinement of the method or stricter control of experimental variables¹⁹

Robustness:

Denotes the capacity of a system or method to retain accuracy and reliability under varying conditions or disturbances. In scientific research, it ensures that outcomes remain valid despite minor parameter shifts or environmental changes, thereby reinforcing the credibility of experimental conclusions^20-25.

Solution Stability:

The stability of solution containing Zinc and Manganese in test and standard solutions was assessed over 24hours at room temperature.

RESULT AND DISCUSSION:

The analytical methods for the quantification of Zinc and Manganese in Multivitamin and Multimineral Syrup were validated by assessing key parameters including specificity, linearity, range, accuracy, precision and solution stability.

Specificity:

The blank, placebo, standard, and test solutions were analyzed to evaluate matrix interference. Absorbance in blank and placebo solutions was <5.0% of the standard, indicating no significant interference^26-28

Linearity:

It was evaluated by analysing multiple concentrations of Zinc and Manganese standards. Zinc standard solutions were prepared at concentrations from 0.2 to 0.6ppm, while Manganese standards were prepared from 0.50 to 1.50ppm. The calibration curves established robust linear relationships with correlation coefficients (R²) of 0.9989 for Zinc and 0.9996 for Manganese.^29-30

The linearity parameters and concentration rang

es for both Zinc and Manganese are presented in Table 1, Table 2 and Table 3. Concentration vs Absorbance for Zinc and Manganese were presented in Figure 1 and Figure 2. These findings indicate that the developed methods are suitable for accurate quantitative analysis of Zinc and Manganese in oral liquids.

Table 1- Linearity data for Zinc

Concentration (mg/mL)	Absorbance
0.1994	0.151
0.2991	0.243
0.3988	0.336
0.4985	0.422
0.5982	0.507

Table 2- Linearity data for Manganese

Concentration (mg/mL)	Absorbance
0.5000	0.128
0.7500	0.194
1.0000	0.258
1.2500	0.316
1.5000	0.381

Table 3- Linearity Parameters of Zinc and Manganese

Parameter	Zinc	Manganese
Linearity range	0.2–0.6ppm	0.50–1.50ppm
R2	0.9989	0.9998
Slope	0.8635	0.2352
Intercept	-0.0136	0.0040
Correlation coefficients	0.9994	0.9996

Figure 1-Graph of Concentration vs Absorbance for Zinc

Figure 2-Graph of Concentration vs Absorbance for Manganese

Precision: Six replicate test solutions were analysed for method precision. For intermediate precision, separately prepared six replicate test solutions were analysed independently by another analyst on next day. The Relative Standard Deviation (RSD) was 1.28% for Zinc and 0.66% for Manganese, confirming acceptable repeatability (RSD<10%). Results for Intermediate Precision are given in Table 4.

Table 4: Results for Method and Intermediate Precision

Element	% Zinc content		% Manganese content
Analyst	Analyst 1	Analyst 2	Analyst 1	Analyst 2
Sample-1	103.00	106.40	106.10	104.50
Sample-2	105.70	106.40	107.00	104.70
Sample-3	104.00	107.60	107.90	105.10
Sample-4	106.00	106.40	107.50	105.10
Sample-5	106.50	106.00	108.00	104.60
Sample-6	104.40	106.60	107.00	106.70
% Average	104.93	106.57	107.25	105.12
% RSD	1.28	0.51	0.66	0.78
% RSD of 12 samples	1.23 %		1.25 %

Accuracy:

Placebo samples were spiked at 50%, 100%, and 150% of nominal concentrations.

Recoveries were:

Zinc: 113.09%, 106.85%, 104.00%

Manganese: 111.17%, 108.79%, 105.76%

The mean recovery per concentration was found well within limits 90-115% and shown in Table 5

Table 5: % Recovery of Zinc and Manganese at 50, 100 and 150%

Level	Sample Name (Recovery sample % / replicate)	% Recovery of Zinc	% Recovery of Manganese
50%	Test solution 1	110.75	109.21
	Test solution 2	114.89	112.57
	Test solution 3	112.62	111.73
	Average	112.75	111.17
	% RSD	2.30	1.57
100%	Test solution 1	108.88	108.79
	Test solution 2	107.24	109.21
	Test solution 3	104.44	108.37
	Average	106.85	108.79
	% RSD	2.10	0.39
150%	Test solution 1	102.18	105.57
	Test solution 2	104.67	105.57
	Test solution 3	105.14	106.13
	Average	104.00	105.76
	% RSD	1.53	0.31

Robustness:

Robustness was performed by carrying out the experiment at low, medium and High sensitivity of detector and percentage was determined on five replicates of sample. Results of robustness are given in Table 6.

Table 6: % Of Zinc and Manganese at Low, Medium and High Sensitivity of detector

Sensitivity	% of Zinc	% of Manganese
Low Sensitivity	109.30%	96.02%
Medium Sensitivity	106.92%	98.92%
High Sensitivity	109.90%	101.0%

Solution Stability:

The stability of test and standard solutions was assessed over 24hours at room temperature. No significant changes were observed in absorbance or calculated concentration during the evaluation period, indicating the solutions remained stable for at least 24 hours under the tested conditions.

CONCLUSION:

The present study describes an analytical method for the simultaneous determination of Zinc and Manganese in multivitamin and multimineral syrup using Atomic Absorption Spectrophotometry (AAS). To provide a clear framework of the method’s performance, the validation results are organized and presented in tables. The data demonstrate that the developed method meets all acceptance criteria of validation outlined in ICH Q2 (R2). Validation of method provided a high degree of reassurance that the stated method will consistently provide accurate results that evaluate a product against its determined specifications and quality attributes. From the above assessment it is concluded that, the method found to be specific, linear, precise and accurate can be used for the routine analysis and stability studies.

ACKNOWLEDGEMENTS:

We sincerely thank everyone who contributed to the successful completion of this research paper on the analytical method for estimating Zinc and Manganese using Atomic Absorption Spectrophotometry. We are especially grateful to Zuventus Healthcare Ltd. for providing the essential resources, facilities, and infrastructure that made this research possible. We also acknowledge with deep appreciation all those who supported this work, directly or indirectly. Your combined efforts have been vital to its success, and we truly value your contributions.

AUTHOR CONTRIBUTIONS:

S.K was responsible for the study's conception, design, and data collection. Data interpretation by V. G., and B. J. The article's crucial intellectual ideas have been revised and written G.K, S.K. and S.P.

DATA AVAILABILITY:

Data will be made available on request.

COMPETING INTERESTS:

The authors declare no conficts of interest related to this work.

REFERENCES:

1. Vieira BM, de Mello e Silva GN, Silva MI. Nutritional elements II: vitamins and minerals. In: Fundamentals of drug and non-drug interactions: physiopathological perspectives and clinical approaches. Cham: Springer Nature Switzerland; 2025. p. 57–86.

2. Islam MR, Akash S, Jony MH, Alam MN, Nowrin FT, Rahman MM, et al. Exploring the potential function of trace elements in human health: a therapeutic perspective. Mol Cell Biochem. 2023; 478(10): 2141–2171.

3. Bailey RL, Carmel R, Green R, Pfeiffer CM, Cogswell ME, Osterloh JD, et al. Monitoring of vitamin B-12 nutritional status in the United States by using plasma methylmalonic acid and serum vitamin B-12. Am J Clin Nutr. 2011;94(2):552–561.

4. Aschner JL, Aschner M. Nutritional aspects of manganese homeostasis. Mol Aspects Med. 2005; 26(4–5): 353–362.

5. Prasad AS. Zinc in human health: effect of zinc on immune cells. Mol Med. 2008; 14(5–6): 353–357.

6. Barnett JB, Dao MC, Hamer DH, Kandel R, Brandeis G, Wu D, et al. Effect of zinc supplementation on serum zinc concentration and T cell proliferation in nursing home elderly: a randomized, double-blind, placebo-controlled trial. Am J Clin Nutr. 2016; 103(3): 942–951.

7. Panel on Micronutrients. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington (DC): National Academies Press; 2002.

8. Nriagu J. Zinc toxicity in humans. Ann Arbor (MI): School of Public Health, University of Michigan; 2007.

9. Santamaria AB. Manganese exposure, essentiality and toxicity. Indian J Med Res. 2008;128(4): 484–500.

10. Farag MA, Hamouda S, Gomaa S, Agboluaje AA, Hariri MLM, Yousof SM. Dietary micronutrients from zygote to senility: updated review of minerals’ role and orchestration in human nutrition throughout life cycle with sex differences. Nutrients. 2021; 13(11): 3740.

11. Agnew UM, Slesinger TL. Zinc toxicity. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023.

12. Kicińska A, Kowalczyk M. Health risks from heavy metals in cosmetic products available in the online consumer market. Sci Rep. 2025;15(1): 316.

13. Evans EH, Pisonero J, Smith CM, Taylor RN. Atomic spectrometry update: review of advances in atomic spectrometry and related techniques. J Anal At Spectrom. 2020;35(5):830–851.

14. United States Pharmacopeial Convention. United States pharmacopeia and national formulary (USP 43–NF 38). Rockville (MD): United States Pharmacopeial Convention; 2020.

15. Borman P, Elder D. Q2 (R1) validation of analytical procedures: text and methodology. In: ICH quality guidelines: an implementation guide. 2017. p. 127–166.

16. Epshtein NA. Validation of the specificity of chromatographic methods: key points and practical recommendations. Pharm Chem J. 2022; 56(5): 702–711.

17. Kadam GM. Development and validation of analytical method for assay and related substances test by liquid chromatographic technique. In: Chief Editor, editor. Book title. Chapter 4. p. 57.

18. Bulska E, Ruszczyńska A. Analytical techniques for trace element determination. Phys Sci Rev. 2017; 2(5): 20178002.

19. McAlinden C, Khadka J, Pesudovs K. Precision (repeatability and reproducibility) studies and sample-size calculation. J Cataract Refract Surg. 2015;41(12):2598–2604.

20. Omoteso OA, Milne M, Aucamp M. The validation of a simple, robust, stability-indicating RP-HPLC method for the simultaneous detection of lamivudine, tenofovir disoproxil fumarate, and dolutegravir sodium in bulk material and pharmaceutical formulations. Int J Anal Chem. 2022; 2022: 3510277.

21. Nishant Sarode, G. S. Chhabra, Shailesh Luhar, Anil Jadhav. Development and Validation of RP-HPLC Method for the Estimation of Montelukast Sodium in Bulk and In Tablet Dosage Form. Research J. Science and Tech. 2011; 3(5): 257-260.

22. Nachiket S. Dighe, Ganesh S. Shinde, Jyoti. J. Vikhe. Simultaneous Estimation, Validation and Force Degradation Study of Metformin Hydrochloride and Empagliflozin by RP-HPLC Method. Research J. Science and Tech. 2019; 11(2):135-147.

23. P. B. Jadhav, S. G. Bhokare, M. N. Madibone. Development and Validation of an RP-HPLC Method for Pamabrom in Bulk and Pharmaceutical Dosage Form. Research J. Science and Tech. 2019; 11(3): 179-182.

24. Akash Shelke, Someshwar Mankar, Mahesh Kolhe. Development and Validation of RP-HPLC Method for estimation of Secnidazole in API and Pharmaceutical Dosage Form. Research Journal of Science and Technology. 2021; 13(2): 100-4.

25. Bhagyashree Tayade, Vikram Gharge, Balasaheb Jadhav, Anant Patil, Chetan Parde, Shraddha Patil. Critical Parameters affecting Stability of APIs and Drug Products: A Review. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 41-0. doi: 10.52711/2231-5675.2026.00007

26. Darshini H B, Mounika, Rama Bukka, Abdul Naim. A High-Precision RP–HPLC Method for Ondansetron Hydrochloride: Development, Optimization and Validation. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 29-3. doi: 10.52711/2231-5675.2026.00005

27. Prachi Rajesh Patil, Javesh K. Patil. Analytical method development and validation for Simultaneous Estimation of Rifampicin and Levofloxacin in Bulk and Synthetic Mixtures Using Validated RP- HPLC Technique. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 21-8. doi: 10.52711/2231-5675.2026.00004

28. Darshini H B, Mounika, Rama Bukka, Abdul Naim. A High-Precision RP–HPLC Method for Ondansetron Hydrochloride: Development, Optimization and Validation. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 29-3. doi: 10.52711/2231-5675.2026.00005

29. Kailash Pati Pandey, K Saravanan. Method Development and Validation for the Assay of the Anticoagulant dosage form by HPLC. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 9-3. doi: 10.52711/2231-5675.2026.00002

30. Laxman A. Kawale, Mayuri V. Gophane, Surabhi H. Patil, Vandana S. Nade. Analytical Quality by Design-Based RP-HPLC Method for Simultaneous Estimation of Lobeglitazone sulfate and Glimeperide in Pharmaceutical Formulations. Asian Journal of Pharmaceutical Analysis. 2026; 16(1): 1-8. doi: 10.52711/2231-5675.2026.00001

Received on 13.03.2026 Revised on 30.03.2026

Accepted on 15.04.2026 Published on 16.04.2026

Available online from April 18, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(2):99-104.

DOI: 10.52711/2231-5675.2026.00014

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.