Quantifying Total, Bound and Free Dextran in Iron Dextran:

A Multi-Method Approach with Dialysis, Gel Filtration Chromatography and HPLC

 

 

ABSTRACT:

Conventional carbohydrate assays often fail to distinguish between dextran that is free in solution and dextran that is covalently or coordinatively bound to iron. Therefore, a multi-method analytical strategy is needed to selectively quantify both fractions. In this study, we developed and validated an approach combining dialysis-based ultrafiltration, gel permeation chromatography (GPC), and high-performance liquid chromatography (HPLC) with refractive index (RI) detection to separate, identify, and quantify free and bound dextran in an Iron Dextran formulation. This method enables comprehensive dextran profiling and supports regulatory-compliant characterization of complex injectable products. Materials and Methods: In first step bound and free dextran was separated from Iron dextran using ultracentrifugal filters with a 10kDa molecular weight cutoff. The separation was monitored by gel permeation chromatography (GPC) using Ultrahydrogel columns (1000Å and 120Å, 7.8mm x 300mm I.D., 12μm particle size) connected in series. The mobile phase consisted of 40mM sodium phosphate buffer (pH 7.0) with 0.02% sodium azide, and detection was performed using a refractive index (RI) detector at a flow rate of 0.5mL/min. In second step Total and bound dextran were hydrolysed to yield glucose, which was then quantified using a validated reverse-phase high-performance liquid chromatography (RP-HPLC) method. The RP-HPLC analysis was performed on a SUGAR SH1011 column (8.0 x 300mm I.D., 6 μm particle size) using water as the mobile phase at a flow rate of 0.6mL/min, with RI detection. Conclusion: This integrated methodology enables the selective and quantitative determination of bound and free dextran components in Iron Dextran formulations. The results showed total dextran content of 60-70%, with 14-18% bound to the iron core and 40-50% in the free form. The synergy of these methods provides a comprehensive understanding of iron dextran's complex structure, highlighting the value of a multi-method approach in pharmaceutical analysis.

 

 

 

 

INTRODUCTION:

Iron dextran, which is also known as, INFeD, is an emerging innovative Iron complex, which comprising a polynuclear Iron (III)-oxyhydroxide core, encased in a carbohydrate dextran matrix, it facilitates the targeted and controlled delivery of iron to specific tissues. The dextran shell's properties^1-4 play a crucial role in determining the complex's stability and iron release rate. The type and amount of total, bound and unbound dextrans in iron dextrans can affect the particle size, surface characteristics, and in vivo performance of the iron dextran. The dextran portion of the iron dextran complex, and how it's bound to the iron core, are important factors in the overall characteristics of the iron dextran.

 

This complex is directed by I.V. route and shows promising results in the handling of Iron-deficiency anaemia. Various trials have revealed that intravenously directed Iron dextran quickly progresses haemoglobin levels and restores low iron levels in a range of people suffering from iron-deficiency anemia, Furthermore, the count includes individuals with inflammatory bowel disease, heavy uterine bleeding, postpartum iron deficiency anemia, or chronic kidney disease. Anemia remains a major health issue, affecting millions of people in both industrialized and developing countries^5-16. As per the World Health Organization's (WHO) estimates, anaemia impacts around 1.62 billion individuals worldwide, accounting for roughly 25% of the global population. Iron deficiency, the cause of anaemia is related to a number of coexisting disorders. For example, Iron-deficiency anaemia can strike people with chronic blood loss-related diseases (such as inflammatory bowel disease or severe uterine bleeding), postpartum women and chronic kidney disease patients^17-24. In some people with chronic illnesses such as those with inflammatory bowel disease or chronic renal disease anaemia may also be caused by factors other than iron shortage, such as inhibition or suppression of erythropoiesis^21-23. The best indicator of anaemia is normal haemoglobin concentrations, which change with age, sex, physiological changes and other variables.

 

According to the World Health Organisation, the haemoglobin values for defining anaemia for pregnant women was 11g/dL^1,9, for non-pregnant women it was 12g/dL, and in case males it was 13g/Dl. In general, ferritin values below 15mg/L1 (or 100mg/L or greater in individuals with chronic illness) are considered depleted iron reserves^23-26 and transferrin saturations <16%^1,23 (<20% in individuals with chronic illness)^24–26. Anaemia due to iron shortage can cause weariness, headaches, dizziness, palpitations, dyspnoea and impaired cognitive function^1,9,20,27. The patient's capacity to work, perform physically and have a positive health-related quality of life (HR-QOL) may all be affected by these symptoms^9,18,23. Iron-deficiency anaemia is linked to mortality and co-morbidity^2,20. The diagnosis, management, and substitution of iron or oral iron preparations are all necessary in the management of iron-deficiency anemia^1,9,22.

 

HPLC has been an extensively employed procedure in the field of pharmaceuticals for the analysis of various chemical compounds. Specially, Dialysis procedure⁴, RP-HPLC and GPC technique^34-35 has shown a promising result in the determination of total and bound dextran in Iron dextran drug substances^29-33. This technique could potentially be applied to the analysis of bound and unbound dextran in Iron Dextran. This research aims to explore the application of Reverse Phase HPLC for the quantitative estimation of bound and unbound dextran in Iron dextran drug material.

 

MATERIALS AND METHODS:

Materials:

HPLC-grade D-glucose (99.80% assay purity) was acquired from Sigma-Aldrich (USA). For method development and validation, a lyophilized sample of the iron dextran drug product, INFeD, was utilized. Hydrochloric acid (37%, A.R. grade) was sourced from Merck (India). Ultrapure water for all aqueous preparations was generated using a Milli-Q Plus purification system (Millipore). All weighing and volumetric measurements were conducted using a Mettler XP-26 electronic microbalance and Biosystem micropipettes (10–100μL and 100–1000μL), both obtained from Merck (Mumbai, India).

 

Instrumentation and Chromatographic Conditions:

Dialysis Procedure To separate free and bound dextran, a dialysis-like approach was applied using centrifugal ultrafiltration. Iron dextran solution was transferred into a 10kDa MWCO filter device and centrifuged at 5000 rpm for 20minutes. The filtrate (containing unbound dextran) was collected. Subsequently, Milli-Q water was added to the retentate, and the centrifugation process was repeated to enhance washing. The retentate, presumed to contain only Iron-bound dextran, was collected separately. This procedure was designed to remove free (unbound) dextran from the formulation. The efficacy of removal was confirmed by analyzing the filtrate using GPC; absence of free dextran peaks indicated successful separation (refer to Figure 1). Gel permeation chromatography (GPC) analyses were performed to monitor the absence of unbound dextran by using a Waters Alliance 2695 separation module coupled with a 2414 refractive index (RI) detector.

 

Figure 1: Chromatogram (a) and (b) obtained from before dialysis and after dialysis respectively.

 

Data acquisition and processing were carried out using Empower 3.0 software. Two columns—Ultrahydrogel 1000Å and 120 Å (7.8mm × 300mm I.D., 12µm particle size)—were connected in series. The mobile phase was 40 mM sodium phosphate buffer (pH 7.0) with 0.02% sodium azide, delivered at a flow rate of 0.5mL/min. The column oven temperature was maintained at 40°C, and the injection volume was 30µL. The system was equilibrated with the mobile phase for 3hours prior to sample injection to stabilize the baseline.

 

Quantitative Determination Total, Bound and Free dextran in Iron Dextran by using HPLC-RI Detector: Following acid hydrolysis of the retentate and original Iron Dextran formulation, released monosaccharides (primarily glucose) were quantified using HPLC-RI. Glucose was used as a marker for total dextran content. This result confirmed that the hydrolysis procedure was robust and suitable for quantitative release of sugar monomers from the polysaccharide backbone and batch data as shown in Table 1.

 

Table 1: Total, Bound and Free Dextran in Iron Dextran in samples

Sample	% Total Dextran	% Bound Dextran	% Free Dextran
Sample-1	68.4	14.4	49.1
Sample-2	68.1	16.7	45.2
Sample-3	68.4	17.4	42.6

 

Instrumentation:

Chromatographic analysis was conducted using a Waters Alliance 2695 HPLC system equipped with a 2414 refractive index (RI) detector. Data acquisition and processing were performed using Empower 3.0 software. Separation was achieved with a Shodex Sugar SH1011 column (8.0 × 300mm, 6.0µm) maintained at a temperature of 40°C. Milli Q water was used as a Mobile phase and diluent. The mobile phase delivered at a flow rate of 0.6mL/min, with an injection volume of 30μL. The sample cooler was held at 10°C throughout the analysis. Prior to sample injection, the column and detector were equilibrated with the mobile phase for 3 hours to ensure baseline stability. Chromatograms were recorded, and the glucose peak was integrated for quantitative analysis.

 

Standard Preparation: Weighed accurately, 280mg of glucose standard was transferred into a 100mL volumetric flask. The addition of 10.0mL of diluent was followed by sonication for 2minutes to ensure dissolution, which was then brought to volume with diluent and mixed.

 

Sample Preparation:

Weighed about 10mg of Iron dextran colloid drug sample and Iron-bound dextran sample into 50mL volumetric flask individually containing 2mL water and add 5N HCl solution, closed with lid. Heated the solution at 80°C temperature for 3.0hr, brought back to room temperature, The volume was made up to the mark with diluent, followed by mixing.

 

RESULTS:

Method Validation:

The method was developed and validated according to the guidelines set by the International Council for Harmonisation (ICH). Parameters such as System Suitability, specificity, linearity, precision and accuracy were evaluated.

 

Chromatographic System Suitability:

To ensure the system's proper functioning, system suitability tests were performed. The system appropriateness was taken into consideration when preparing standardly. Measurements were made of all crucial factors, such as theoretical plate number and peak tailing. The RSD of glucose peak areas was measured by injecting a standard solution six times in replicate. as presented in Table 2. The chromatographic procedure demonstrated consistent glucose retention times (RT) ≈ 12.36min (see Figure 2). The system, technique, and column performance were all covered under these system suitability characteristics.

 

 

Table 2: System suitability of glucose

S. No.	Sample ID	RT	Area	USP Plate Count	USP Tailing
1	Standard_01	12.361	5639166	6894	1.2
2	Standard_02	12.361	5639124	6877	1.2
3	Standard_03	12.365	5644727	6848	1.2
4	Standard_04	12.365	5627862	6867	1.2
5	Standard_05	12.362	5642513	6872	1.2
6	Standard_06	5637944	6875	1.2
Avg	—	12.363	5638556	—	—
SD	—	0.002	5816.7	—	—
% RSD	—	0	0.1	—	—

 

 

Figure 2: Chromatograms obtained from (a) blank, (b) standard glucose solution, and (c) iron dextran sample solution.

 

Precision and Accuracy:

Precision was assessed using a sample preparation strategy that involved preparing six replicates, which were then analyzed through the proposed HPLC method. To evaluate intermediate precision, the analysis was conducted again by a second analyst on a different day, utilizing a separate chromatographic column and instrument. The relative standard deviation (RSD) of the glucose standard area, along with the calculated percentage of dextran, served as indicators of method precision. The results indicated excellent repeatability and intermediate precision, with % RSD values remaining comfortably within acceptable limits, as detailed in Table 3.

 

Table 3: Precision data for Dextran determination

Sample Prep	Area	% Dextran
1	4970289	61.2
2	5006561	61.7
3	4987643	61.5
4	4974125	61.3
5	5017072	61.8
6	4989097	61.5
Average	—	61.5
SD	—	0.23
%RSD	—	0.4

To evaluate the accuracy of the developed method, recovery studies were conducted at three distinct sample concentration levels: 50%, 100%, and 150%, which correspond to 0.20mg/mL, 0.40mg/mL, and 0.60mg/mL of dextran, respectively. At each concentration level, the percentage of dextran recovered was calculated. The average dextran content obtained at these concentrations was 61.1%, 61.6%, and 60.3%, demonstrating high accuracy and minimal interference from the matrix. Recovery values ranged from 98.0% to 100.7%, as outlined in Table 4.

 

Table 4: Accuracy data

Sample	Conc. (mg/mL)	Area	% Dextran	% Recovery	% RSD
50% level_01	0.2	2477677	61.1	99.3	0
50% level_02	0.2	2479376	61.1	99.3	0
50% level_03	0.2	2477593	61.1	99.3	0
100% level_01	0.4	4994252	61.5	100	0.4
100% level_02	0.4	5021366	61.9	100.7	0.4
100% level_03	0.4	4992104	61.5	100	0.4
150% level_01	0.6	7334474	60.3	98	0
150% level_02	0.6	7340861	60.3	98	0
150% level_03	0.6	7328623	60.3	98	0

 

Specificity:

The specificity of the method was determined by injecting a blank solution, sample and Dextran to observe no interference with the retention time of Glucose.

 

Linearity:

The analysis of the data was done using the linear regression method. The linearity of the method was evaluated by plotting the peak area of the standard compound against its concentration, and the resulting calibration curve was characterized by its regression equation, correlation coefficient, and Y-intercept bias. Response for Glucose was linear within the concentration range of 70, 140, 210, 280, 350 and 420 ppm. The results, shown in Figure 3, demonstrate excellent linearity with a correlation coefficient of 0.9999.

 

Figure 3: Linearity of glucose.

 

RESULTS AND DISCUSSION:

The analytical methods were evaluated for precision, recovery, and specificity. The intra-day precision (repeatability) of the HPLC method was below 2% RSD for dextran quantification. Recovery studies conducted on spiked samples confirmed an average recovery of 97–102%, indicating method accuracy. The system suitability criteria (baseline stability, retention time repeatability, and peak shape) were consistently met across all runs.

 

This integrated approach using dialysis, GPC, and HPLC-RI analysis offers a comprehensive, reproducible, and specific means of differentiating and quantifying free versus bound dextran in Iron Dextran formulations. The ultrafiltration strategy served as a reliable physical method for separation, while the chromatographic techniques provided molecular and quantitative characterization of the dextran fractions. The method is suitable for both routine quality control and regulatory submissions requiring precise dextran content specification in iron dextran complex formulations.

 

CONCLUSION:

A combined approach employing dialysis, gel permeation chromatography, and HPLC-RI detection was successfully developed for the selective quantification of free and bound dextran in Iron Dextran formulations. The dialysis method effectively separated unbound dextran, as confirmed by GPC analysis. Hydrolysis of the retained complex and subsequent HPLC quantification allowed accurate measurement of dextran content based on glucose release. The method demonstrated excellent specificity, precision, and recovery, making it suitable for routine quality control, formulation development, and regulatory compliance in biopharmaceutical settings.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Department of Chemistry, Acharya Nagarjuna University, for their support and guidance, which played a significant role in the successful completion of this research project.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

 

ABBREVIATIONS:

RP-HPLC: Reverse phase High performance liquid chromatography; RI detector: Refractive Index detector; HCl: Hydrochloric acid; RT: Retention time; RSD: Relative standard deviation; SD: Standard deviation.

 

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Received on 20.05.2025      Revised on 23.09.2025 Accepted on 03.12.2025      Published on 05.06.2026 Available online from June 06, 2026 Research J. Pharmacy and Technology. 2026;19(6):2563-2568. DOI: 10.52711/0974-360X.2026.00367 © RJPT All right reserved
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