Simultaneous Quantification of Calcium and Magnesium in Pharmaceutical Ingredients and supplements (Injections, and Tablets) using Carboxyl Moiety Stationary Phase HPLC Column with Refractive Index detector

Nagavardhana Reddy Vanga¹, Venkata Ratnam K¹*, Kumarswamy Ummiti²,

Venkata Nadh Ratnakaram¹

¹Industrial Chemical Product Development and Analysis Centre, Department of Chemistry,

GITAM School of Science, GITAM Deemed to be University – Bengaluru Campus, Karnataka – 561203, India.

²Department of Chemistry, College of Engineering, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, India – 522502.

*Corresponding Author E-mail: vratnam2004@gmail.com

ABSTRACT:

Calcium (Ca) and magnesium (Mg) are two essential minerals that help keep bones strong and healthy. Our bodies do not create calcium and magnesium naturally, so we must receive them from food or supplements. Ca and Mg supplements in various dosage forms, such as tablets and injectables, are available to prevent deficiency. It is essential for determining the levels of Ca and Mg in drug substances and pharmaceutical compounds. Methodological approaches such as inductively coupled plasma mass spectrometry (ICP-MS), ion chromatography (IC), atomic absorption spectroscopy (AAS), capillary electrophoresis (CE), and titration are available for estimating Ca and Mg. The current research devised a novel isocratic reverse-phase HPLC method, an alternative approach to ICP-MS. It uses 0.7% v/v formic acid as the eluent, a carboxyl functional group-packed HPLC column, and a refractive index detector (RID) to determine Ca and Mg content. The method was validated as per regulatory requirements. The method has the optimal sensitivity (quantification threshold: 2 µg/ mL and detection threshold: 0.6µg/mL), is specific and inexpensive, and is thus well suited for the detection and quantification of Ca and Mg in drug substances and in supplements, including injections and tablets.

KEYWORDS: Calcium (Ca), Magnesium (Mg), Supplements, Assay, Refractive index detector (RID), Carboxyl functional group packed HPLC column.

INTRODUCTION:

Calcium (Ca) and magnesium (Mg) are important for the bones, blood-clotting systems, heart, and nerves to work. ¹ Mg deficiency has been related to disorders including osteoporosis, hypertension, cardiac problems, genetic heart disease, obesity, and stroke.^2,3 Ca supplements treat and prevent low calcium levels and bone problems that arise, such as muscle cramps, osteoporosis, rickets, and osteomalacia.^4-10

Ca and Mg deficiency can be prevented in several ways, including the use of tablets or injections. Ca supplements include coral calcium tablets (Ca and Mg), calcium chloride injection (Nelcium), calcium gluconate+calcium lactobionate injection (Ca-Sandoz injection), and so on. Mg supplements include magnesium oxide tablets (RemMag), magnesium sulfate injections (Magneon), and so on. It is required to accurately determine Ca and Mg levels in pharmaceutical compounds.

Ca and Mg levels can be determined using titration¹¹, IC ¹², AAS¹³, CE¹⁴, and ICP-MS¹⁵. The titration method will use edetate sodium as a titrant to determine Ca and Mg. Since titration is done manually, there is a high likelihood that different people will find different endpoints. Due to this reason, regulatory agencies are anticipating stability indicating methods to determine the active components in pharmaceutical products.¹⁷Small research and development labs often have challenges in obtaining IC, AAS, and CE equipment. Furthermore, HPLC is more user-friendly in comparison to the aforementioned devices. Therefore, our objective was to create a highly sensitive HPLC method to identify and quantify the levels of Ca and Mg in pharmaceutical products.

There have been no reports of Ca and Mg levels being determined in supplements using HPLC and RID. In the current study, Ca and Mg concentrations were quantified using an HPLC method connected with a RID. Chromatographic separations were performed using an HPLC column packed with the carboxyl (-COO^-) functional group as the stationary phase. For each sample analysis, the chromatographic run time was 10 minutes. This method was validated by the guidelines of the International Convention on Harmonization.¹⁶

MATERIALS AND METHODS:

Calcium+Magnesium tablets (brand name: Coral Calcium, manufacturer: Premier Nutraceuticals Pvt.Ltd., lot no. PCC1413, expiration date: 09/2026), Calcium chloride injection I.P (brand name: Nelcium, manufacturer: Neon Laboratories Ltd., lot no. 374018, expiration date: 07/2023), Calcium Gluconate+Calcium Lactobionate (brand name: Calcium-Sandoz injection, manufacturer: Sovereign Pharma Pvt Ltd., lot no. 104SPAA2, expiration date: 12/2023), Magnesium oxide tablets 400mg (brand name: RemMag, manufacturer: Proqol Healthcare Pvt.Ltd., lot no. PT596, expiration date: 08/2024), Magnesium sulfate injection I.P (brand name: Magneon, manufacturer: Neon Laboratories Ltd., lot no. KP370073, expiration date: 06/2023) were bought from a pharmacy in India for this research study. Magnesium sulfate heptahydrate (emsure grade), Hydrochloric acid 37% (emsure grade), Sulfuric acid 98% (emsure grade), Formic acid 98-100% (ACS grade), and Calcium chloride dihydrate (emsure grade) were purchased from Avantor, India. Gluconic acid and Lactobionic acid were purchased from BLD pharmatech Ltd (Shanghai, China). The water collected from the water purification system manufactured by Biolab Scientific Limited (model no. BLPS-101, Canada) was utilized for eluent preparation.

All the chromatographic experimentations were done on a Waters HPLC e2695 separation module system equipped with Empower software and a RID (Model 2414, USA). For chromatographic separations, the following method parameters were selected: eluent and diluent -0.7% v/v formic acid in water; detector type- HPLC coupled with refractive Index (RI) detector; column- IC YS-50 (125mm x 4.6mm I.D., Shodex, Japan); injection volume- 10µL; flow rate- 1.1 mL/minute; column oven temperature- 35°C; sample cooler temperature- 25°C; run time- 10min; elution approach- Isocratic; RI detector attenuation (sensitivity)- 64; optical unit temp.- 35°C; polarity- positive (+).

Standard solution:

A standard solution of 100µg/mLof Ca and Mg was prepared by dissolving an appropriate amount of calcium chloride dihydrate and magnesium sulfate heptahydrate in the diluent.

Sample solutions:

Each tablet of Coral Calcium contains 225mg of Ca and 12.85mg of Mg. Each Remmag tablet contains 400mg of magnesium oxide, MgO (equivalent to 241.2mg of Mg). Twenty Coral Calcium and Remmeg tablets were weighed individually, and then the average weight was determined. The tablets were crushed into a fine powder. To determine the Ca and Mg content of the Coral Calcium supplement, tablet powder equivalent to 25mg of Ca and 1.43mg of Mg was transferred into a 250mL volumetric flask. To determine the Mg content of the RemMag sample, tablet powder equivalent to 25mg of Mg was transferred into a 250mL volumetric flask. Following the weighing, 150mL of diluent was added and the mixture was subjected to sonication for 30 minutes, with occasional shaking. Following sonication, placed the sample volumetric flask aside and allowed it to cool down to the ambient temperature. After diluting the solution to the right amount with diluent, mix it well. The solution was subsequently filtered with a 0.45µ PTFE syringe filter (Tisch Scientific, Catalogue No. SPEC17163). Discarded the initial 5mL of filtered solution, then transferred the sample solution into an HPLC vial. The concentration of Ca and Mg in the Coral Calcium sample solution is 100µg/mLand 6µg/mL respectively. The concentration of Mg in the RemMag sample solution is 100µg/mL.

Each mL of Nelcium contains 100mg of calcium chloride dihydrate (equivalent to 27.3mg of Ca). A concentration of 110µg/mL of Ca was achieved by diluting 1mL of Nelcium injectable solution to 250mL with diluent. Each mL of calcium sandoz injection contains 50mg of calcium gluconate (equivalent to 4.66 mg of Ca) and 87.5mg of calcium lactobionate (equivalent to 4.65mg of Ca). Overall Each mL of calcium sandoz injection contains about 9mg of Ca. A concentration of 110µg/mL of Ca was achieved by diluting 3mL of calcium sandoz injection solution to 250 mL with diluent. Each mL of Magneon 50% solution contains 500mg of magnesium sulfate heptahydrate (equivalent to 49.3mg of Mg). A concentration of 100 µg/mL of Mg was achieved by diluting 0.5mL of Magneon 50% solution to 250mL with diluent.

RESULTS AND DISCUSSIONS:

The metal atoms Ca and Mg do not constitute a UV chromophore. As a consequence of this, for Ca and Mg detection, an RID was selected.^17-20 Diverse RID attenuations were examined to obtain optimum Ca and Mg responses while minimizing detector noise. The preferred RID's attenuation was 64. To prevent fluctuations in the refractive index, the RID temperature was kept at 35°C, like the column oven temperature. Metal atoms will be present in cationic form in their salts. Ca and Mg, for example, will be present in a divalent cationic form in all salts, i.e., Ca⁺² and Mg⁺². For chromatographic separations, a Shodex IC YS-50 carboxyl functional group-packed column (125 mm x 4.6mm I.D., Japan) was used. Because of the polar-polar interaction, the carboxyl (COO-) group in the stationary phase will hold on to the cations in the introduced sample. The chloride, gluconate, lactobionate, and sulfate peaks will all elute early in the chromatographic column.

While organic enhancers like methanol and acetonitrile are being added, Ca and Mg start to elute in the dead volume. At first, different amounts of orthophosphoric acid and formic acid were mixed with different amounts of water. In comparison to orthophosphoric acid eluents, formic acid eluents produced more symmetrical Ca and Mg peaks. It substantiates the fixation of formic acid as the eluent. The retention times were reduced and peak shapes of Ca and Mg were improved by increasing the formic acid content in the eluent. The eluent was chosen to be formic acid in water (0.7% v/v). The eluent was injected as a blank to verify any blank interference at the retention time of Ca and Mg. The blank chromatogram [Figure 2] showed two peaks at 2.5 and 3.4 minute retention times. Retention times of Mg and Ca ions are 4.6 and 5.4 minutes, respectively. Column oven temperature had no significant effect on Ca, Mg, excipient, or anion peak separations (which were evaluated in the column oven at 25°C to 45°C). The chloride (RT 17min), gluconate (RT 1.92min), lactobionate (RT 1.86 min), and sulfate (RT 1.6min) peaks are separated from the Ca and Mg peaks in both the sample and standard solutions using the chromatographic parameters indicated in the materials and methods. The RTs were confirmed by injection of solutions of gluconic acid (2.34mg/mL), lactobionic acid (4.3mg/mL), sulfuric acid (160µg/mL), hydrochloric acid (290µg/mL), titanium dioxide (100µg/mL). Titanium dioxide did not display any peaks other than blank peaks. Figure 1 shows a representative HPLC chromatogram of a standard solution containing both Ca and Mg. Figure 3 and Figure 4 show a representative HPLC chromatogram of a calcium sandoz injection and coral calcium tablets sample solutions. These results validated the method's specificity.

The RSD of the Ca and Mg responses from the standard solution for the five replicate injections was used to determine the system appropriateness. The observed RSD was less than 2.0%, indicating that the system is accurate and trustworthy. Peak symmetry and sharpness are determined by the plate count and tailing factor. Both the tailing factor and plate count for the Ca and Mg peaks were less than 2.0 and greater than 1500, indicating that the Ca and Mg peaks are symmetrical and narrow. In the standard solution, optimal resolution (>1.5) was seen between the Ca and Mg peaks. Table 1 shows a summary of the data for system suitability.

Table 1. Summarized data of System suitability data of Magnesium and Calcium

Name	% RSD		Tailing factor		Plate count		Resolution
Name	Limit	EV	Limit	EV	Limit	EV	Limit	EV
Mg⁺²	≤ 2.0%	0.4	≤ 2.0	1.6	≥ 1500	1819	≥ 1.5	NA
Ca⁺²	≤ 2.0%	0.3	≤ 2.0	1.8	≥ 1500	2427	≥ 1.5	1.8

EV-Experimental value

Figure 1. HPLC chromatogram of Calcium chloride dihydrate and Magnesium sulfate heptahydrate standard solution

Figure 2. HPLC chromatogram of Blank (diluent)

Figure 3. HPLC chromatogram of Calcium Sandoz injection sample

Figure 4. HPLC chromatogram of Magnesium sulfate heptahydrate injection sample

The S/N ratio was used to find the DT and QT for Ca and Mg. This was done by injecting several diluted solutions with known concentrations. The obtained DT and QT concentrations for Ca and Mg are 0.6µg/mL and 2µg/mL, respectively. At these concentrations, the S/N ratio is >3 for DT and >10 for QT respectively. To confirm repeatability at the QT level, the QT concentration solution was injected into six replicates in a precision study [Figure 5]. The Ca and Mg responses at LOQ concentration (2µg/mL) had an RSD of less than 5%, indicating that the QT concentration is reliable as well as consistent. A study on accuracy at QT concentration was also carried out by spiking 2µg/mLCa and Mg on diluent. At the QT level, Ca and Mg recovery were found to be 95-105%. These results validated the method's accuracy at the QT concentration (Table 2).To investigate the Ca and Mg linearity, six different concentrations were injected, ranging from 2 µg/mL (QT) to 150µg/mL (150% of the intended standard concentration). The correlation coefficient (r) from the Ca and Mg linearity plots was >0.998, showing that the procedure was linear and that there was an adequate relationship between the Ca and Mg concentrations and peak responses ranging from 2 µg/mL to 150µg/mL. The linearity test results are shown in Table 2. The repeatability (precision) of the method was evaluated by injecting six different samples of Ca and Mg drug products (100µg/mL Ca and Mg) as explained in the section on sample preparation. The RSD of Ca and Mg supplements assay findings from six preparations were used to determine method precision (repeatability). Ca and Mg prescription product assays were positive in the 98.4-102.3% range and the percentage of RSD was found to be within 2.0%. These findings confirmed the method's precision (Table 3). The Ca and Mg accuracy investigation was conducted at three different concentrations ranging from 50µg/mL (50%) to 150µg/mL (150%) of the intended sample concentration. The recovery of Ca and Mg was determined to be between 98.9 and 102.2%. The accuracy of the procedure was demonstrated by these findings. According to the linearity, precision, and accuracy studies, the described analytical method for the quantification of Ca and Mg has a range of 2µg/mL to 150µg/mL. The test procedure's robustness was evaluated by changing chromatographic parameters like the temperature of the column oven (30°C to 40°C), the concentration of formic acid in the eluent (0.6% v/v and 0.8% v/v), and the flow rate of eluent (1.0 and 1.2mL/ min). In each condition, the key quality aspects of the test procedure were evaluated. These included the tailing factor, plate count, resolution between Ca and Mg, and the percentage of RSD of Ca and Mg responses. The fact that all of the purposefully modified experimental conditions did not affect the method's discrimination indicates the method's robustness (Table 5).

Figure 5. HPLC chromatogram of QT concentration-2 µg/mL

Table 2. Summarized data of DT, QT concentration, QT accuracy, and linearity for Mg and Ca

Name	DT conc.(µg/mL)	QT conc. (µg/ mL)	% RSD	% Recovery	Linearity
Name	DT conc.(µg/mL)	QT conc. (µg/ mL)	QT precision	At QT level	Conc, range (µg/mL)	r	Bias at 100%
Mg⁺²	0.6	2	3.5	93.4-100.2%	2-150	0.9999	-0.9
Ca⁺²	0.6	2	1.8	97.5-102.1%	2-150	0.9999	0.7

Table 3. Summarized data of method precision of Magnesium and Calcium in various supplements

Supplement name	Assay range (Mg⁺²)	Assay range (Ca⁺²)	% RSD
RemMag ( n=6)	98.9-101.7%	--	1.1
Magneon (n=6)	98.9 -99.8%	--	0.3
Coral calcium for magnesium (n=6)	98.4-100.4%	--	0.9
Coral calcium for calcium (n=6)	NA	98.5-99.9%	0.5
Nelcium (n=6)	NA	100.9-102.3%	0.3
Calcium sandoz (n=6)	NA	99.5-100.8%	0.5

Table 4. Summarized data of Accuracy study

Supplement name	Accuracy level	Recovery range		% RSD
Supplement name	Accuracy level	Mg⁺²	Ca⁺²	Mg⁺²	Ca⁺²
Magneon	50% (50 µg/mL)	99.1 -101.1%	--	1.0	--
	100% (100 µg/mL)	99.0-100.7%	--	0.9	--
	150% (150 µg/mL)	101.5- 101.8%	--	0.2	--
RemMag	50% (50 µg/mL)	99.9 -101.5%	--	0.8	--
	100% (100 µg/mL)	99.1-100.1%	--	0.5	--
	150% (150 µg/mL )	100.6- 101.1%	--	0.2	0.4
Coral calcium	50% (50 µg/mL)	99.7 -101.5%	98.2 -99.7%	0.9	0.8
	100% (100 µg/mL)	98.9-101.1%	98.2-99.3%	1.1	0.6
	150% (150 µg/mL)	101.2- 101.5%	98.1- 98.6%	0.2	0.3
Nelcium	50% (50 µg/mL)	--	100.7-102.2%	--	0.8
	100% (100 µg/mL)	--	99.9 -100.8%	--	0.5
	150% (150 µg/mL)	--	100.4-100.9%	--	0.3
Calcium Sandoz	50% (50 µg/mL)	--	99.9- 101.2%	--	0.7
	100% (100 µg/mL)	--	99.2 -101.8%	--	1.4
	150% (50 µg/mL)	--	99.0-100.8%	--	1.0

^aRSD - % relative standard deviation of recovery at each level (n=3)

Table 5. Summarized data of Robustness study

Robustness parameter	Retention time		% RSD		TF		Plate count		Resolution
Robustness parameter	Mg⁺²	Ca⁺²	Mg⁺²	Ca⁺²	Mg⁺²	Ca⁺²	Mg⁺²	Ca⁺²	Mg⁺²	Ca⁺²
FR (1.0 mL/ min)	5.282	6.218	0.2	0.5	1.6	1.6	1805	2457	--	1.8
FR (1.2 mL/ min)	4.123	4.851	0.1	0.5	1.6	1.5	1734	2290	--	1.8
Oven temp.(30°C)	4.621	5.453	0.2	0.2	1.6	1.5	1717	2246	--	1.8
Oven temp.(40°C)	4.644	5.452	0.4	0.6	1.6	1.5	1814	2443	--	1.8
FA (0.6%v/v)	4.995	5.925	0.3	0.3	1.7	1.6	1599	2238	--	1.8
FA (0.8%v/v)	4.575	5.375	0.1	0.5	1.6	1.6	1780	2365	--	1.8

FR: Flow rate; FA: Formic acid; TF: Tailing factor

CONCLUSION:

After going through the validation process, it was determined that the approach works effectively with a Ca and Mg concentration of 2-150 µg/mL. It is robust, accurate, and precise in addition to being specific and linear. This approach can detect Ca and Mg at concentrations as low as 0.6 µg/mL and is suitable for quantifying Ca and Mg in all Ca and Mg supplements. The chromatography has a run duration of ten minutes. This method is straightforward to use in QC laboratories for monitoring the Ca and Mg assay in Ca and Mg ingredients and supplements. The reported HPLC approach is an alternative stability-indicating methodology for IC, AAS, CE, and ICP-MS.

ACKNOWLEDGEMENTS:

We thank Mr. Vijayabhanu Prattipati (Managing Director), Pleiades Therapeutics Private Limited, Hyderabad, India, for providing the necessary facilities to perform the work.

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Received on 23.02.2024 Revised on 12.06.2024

Accepted on 23.09.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1244-1249.

DOI: 10.52711/0974-360X.2025.00180

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