Determination of Oxalate in Calcium Gluconate Injection Using a NovaChrom Ion Chromatography Column

Calcium gluconate is an organic calcium salt widely used in food and pharmaceutical products, primarily for calcium supplementation and the treatment of calcium deficiency. During the processes of oxidation, fermentation, and degradation, glucose can produce a small amount of oxalic acid as a byproduct. The combination of oxalic acid with calcium directly reduces the bioavailability of calcium supplements, leading to diminished calcium supplementation efficacy. Excessive oxalic acid may trigger hypocalcemia, resulting in neuromuscular excitability abnormalities and cardiac arrhythmias. These risks are particularly critical for patients receiving intravenous administration, and therefore demand stringent prevention and control measures. Consequently, the oxalic acid content in calcium gluconate serves as one of the important quality indicators for assessing product quality. In the revised draft standard of the Chinese Pharmacopoeia (2025 edition) by the Chinese Pharmacopoeia Commission, the determination of oxalate has been added to the draft standard for calcium gluconate.

In this study, a NovaChrom carbonate-based anion chromatography column(CS-5A-P4, 4.0 × 250mm), was employed for the determination of oxalate in calcium gluconate injection. The testing procedure is straightforward, and the results are stable and fully meet the analytical requirements. This provides a reliable reference for the determination of oxalate in calcium gluconate injection using ion chromatography.

Keywords: ion chromatography column; Chinese Pharmacopoeia; glucose; anion chromatography column; calcium gluconate injection; oxalic acid.

1. Experiment

1.1 Main Instrument and Reagents

Ion chromatograph: Wayeal ion chromatograph equipped with a conductivity detector.

Anion chromatography column: CS-5A-P4, 4.0×250 mm

Anion guard column: HS-5AG, 4×30 mm

Sodium oxalate: electronic grade

A commercial brand of calcium gluconate injection

Disposable syringe (2mL)

Aqueous syringe filter (0.45μm)

System suitability: The theoretical plate number calculated from the oxalate peak shall be no less than 2500, and the tailing factor shall be no greater than 1.2.

1.2 Sample Pretreatment

1.2.1 Reference Solution

Take an appropriate amount of sodium oxalate, accurately weigh it, dissolve and quantitatively dilute with hydrochloric acid solution (1 → 1200) to produce a solution containing approximately 1.5μg of sodium oxalate per 1mL (equivalent to approximately 1μg of oxalate per 1mL).

1.2.2 Test Solution

Take an appropriate amount of this product, accurately weigh it, dissolve and quantitatively dilute with hydrochloric acid solution (1 → 1200) to produce a solution containing approximately 20mg per 1mL.

1.3 Instrument Working Conditions

Chromatographic column temperature: 30 °C

Conductivity cell temperature: 35 °C

Eluent: 5.0mM sodium carbonate + 1.0mM sodium bicarbonate

Flow rate: 1.0mL/min

Injection volume: 50μL

2. Results and Discussion

2.1 Test of Reference Solution

The reference solution after pretreatment according to the sample preparation method described in section 1.2 was injected into the system and analyzed under the instrumental conditions specified in section 1.3. The testing chromatogram is shown in Fig 1, and the test results are presented in Table 1 below.

Fig 1 Chromatogram of the Reference Solution

Table 1 Test Results of the Reference Solution

Note: The theoretical plate number calculated from the oxalate peak was 9842, and the tailing factor was 1.163.

2.2 Test of Sample Solution

The sample solution after pretreatment according to the sample preparation method described in section 1.2 was injected into the system and analyzed under the instrumental conditions specified in section 1.3. The testing chromatogram is shown in Fig 2, and the test results are presented in Table 2.

Fig 2 Chromatogram of the Sample Solution

Table 2 Test Results of the Sample Solution

Note: The theoretical plate number calculated from the oxalate peak was 8827, and the tailing factor was 1.119.

2.3 Repeatability Test

Fig 3 Chromatograms of the Reference Solution for 6 Consecutive Injections

Fig 4 Chromatograms of the Sample Solution for 6 Consecutive Injections

Table 3 Test Data

Note: The RSD values of retention time for both the sample solution and the reference solution ranged from 0.027% to 0.113%, and the RSD values of peak area ranged from 0.426% to 0.505%.

3. Conclusion

The NovaChrom carbonate-based anion chromatography column (CS-5A-P4, 4.0 × 250mm) coupled with the Wayeal ion chromatograph was successfully applied to the determination of oxalate in calcium gluconate injection. The method demonstrated high accuracy, good resolution, stable repeatability, and high sensitivity. All results met the system suitability criteria specified in the Chinese Pharmacopoeia, with theoretical plate numbers exceeding 2500 and tailing factors below 1.2 for the oxalate peak.