Determination of Sodium, Potassium, and Magnesium as Sulfate Salts in Oral Preparations Using Ion Chromatography and Conductivity Detection

Abstract

An ion chromatography technique with conductivity detection was selected as an analytical tool for the simultaneous indirect determination of sodium sulfate, potassium sulfate, and magnesium sulfate via their respective cations. The method was developed and validated for the quantitative assay of the inorganic salts under study in oral pharmaceutical dosage forms. Chromatographic separation was achieved on a Dionex^®IonPac^® CS16 column (250 × 5 mm) column using the gradient elution method. A mobile phase-A consisting of methane sulfonic acid (6.7%, v/v) in Milli-Q water, which is used together with Milli-Q water, was used as a mobile Phase-B. The flow rate was 1.2 mL/min. The retention times of sodium, potassium, and magnesium as sulfates were 7.8, 12.8, and 16.2 min, respectively. The method was validated according to ICH guidelines and showed good linearity and accuracy results within concentration ranges of 80.0–240.0, 20.0–60.0, and 4.5–13.5 ppm for sodium, potassium, and magnesium as sulfates, respectively. The relative standard deviation results for intra- and inter-day precision were less than 1.0%. The method was applied successfully for determination of the analytes under study in their mixed pharmaceutical oral solution and found suitable for their routine and stability analysis.

1. Introduction

Ion chromatography (IC) is one of the most established techniques where variable ionic species can be analyzed. Several developments have been made to IC during the past years, as this technique was approved and utilized widely by several regulatory entities, such as the British Pharmacopeia (BP), United-States Pharmacopeia (USP), Association of Official Agricultural Chemists (AOAC), and others [1]. The low-cost, repeatability, and full automation of IC account for its higher reliability during routine limit tests and regular monitoring of different inorganic analytes [2]. Several factors affect the reliability of IC methodologies, including sample preparation, detectors in use, stationary phase mode, and even type of the analytes [3]. Therefore, IC can be easily modulated for better selectivity and specificity.

Although spectroscopic techniques like inductively coupled plasma coupled with optical emission spectroscopy or mass spectroscopy (ICP-OES and ICP-MS) and atomic absorption spectroscopy (AAS) have strong competition with IC on metal cations determination, however, IC has higher capability. IC has the advantage of the capability to distinguish between different oxidation states of metals (e.g., Fe⁺²/Fe⁺³), and hence better speciation. Moreover, conductivity detection when combined with IC is now a well-established technique which is cheap to buy, low operating costs, easy to use, sensitive enough for several purposes, and easy to couple with chromatography than direct estimation of analytes with spectroscopic techniques. This means that IC when coupled with conductivity detection can determine cations in presence of other different species without interferences and at higher sensitivities than spectroscopic methods [4].

Sodium sulfate is a white crystalline and hygroscopic powder [5], which is widely used in many industries, like glass, paper, textile, chemical, and pharmaceutical industries. Potassium sulfate is a non-flammable white crystalline salt which cannot be found naturally from earth. Magnesium sulfate is a small colorless and extremely hygroscopic crystal, that is also known as Epsom salt [5]. Both magnesium and potassium sulfates are also used in many industries, like agriculture, food, and pharmaceutical industries.

The combination of sodium sulfate, potassium sulfate, and magnesium sulfate is used as oral preparations to evacuate the colon (bowel) before a colonoscopy [6]. This oral sulfate combination increases colon motility, resulting in diarrhea which can be beneficiary in colon preparation before performing endoscopy or during toxicity emergency [7]. Such low volume oral sulfate solution (OSS) was studied in detail and proved to be a cost-effective and reasonably tolerated solution [8,9,10]. Several tablet and oral solution laxative preparations are being formulated currently for these combined sulfates.

Analytical chemists utilize combustion ion chromatography (CIC) as a tool to determine chloride and sulfate from sebacic acid type of matrices as it is very difficult to dissolve in water. Though, CIC eases handling of organic matrices for its halides estimation, procuring, and operational costs are high and also the methods are tedious. Similarly, cations are estimated using Atomic Absorption Spectroscopy (AAS) or Inductively Coupled Plasma—Optical Emission Spectroscopy or Mass Spectrometry analysis. However, it is an altogether different set up and has its own limitations.

A variety of methods have been developed for analyzing such mineral inorganic cations in different matrices, including popularly used atomic spectroscopic methods such as atomic absorption spectroscopy (AAS), inductively coupled plasma–mass spectrometry (ICP-MS) [11], atomic emission spectrometry (ICP-AES), and optical emission spectrometry (ICP-OES), as well as capillary electrophoresis (CE). However, some of these methods suffer from spectral and chemical interferences, asynchronous determination of mixed cations, laborious and prolonged procedures for sample preparation, as well as utilization of toxic concentrated acid and other reagents. Capillary electrophoresis has limits of poor reproducibility of migration times and peak areas, and moderate sensitivity. Due to the sensitivity, stability of the separation system, good selectivity, and capacity of multi-element analysis in a single run, ion chromatography coupled with conductivity detection (IC-CD) has become the method of choice for the separation and determination of multiple cations and anions in various sample matrices. This powerful and reliable technique has been widely used for analysis of Na⁺, K⁺, Mg²⁺, and anions in biological samples, food, biodiesel, oil, plant extract, and water, particularly at the level of trace concentrations

The method described in the present article displays advantages in its simplicity and accuracy using conventional ion chromatography. Both anions and cations can be precisely analyzed using methods illustrated here in the article, with ease and economically.

A literature survey reveals that some analytical techniques had been established for the determination of sodium, potassium, and magnesium as sulfates. The majority of these techniques used either the ICP-OES and ICP-MS [12,13] or AAS [11] in different matrices. Few methods were reported based on the IC technique for determination of the analytes under study in different matrices like river-water [14] and biodiesel [15]. To the best of our knowledge, no method based on the IC technique was reported for the determination of the analytes under study in their pharmaceutical oral preparations. Thus, the main purpose of this study was to develop a fast, selective, and sensitive analytical method based on the IC technique for the determination for the assay of sodium, potassium, and magnesium sulfates in their oral solution preparations.

2. Materials and Methods

2.1. Materials

Sodium, potassium, and magnesium standards for ion chromatography (IC, 1000 ppm) were procured from Sigma-Aldrich (St. Louis, MA, USA). Analytical grades from sodium sulfate, potassium sulfate, and magnesium sulfate were purchased from Sigma-Aldrich (Burlington, MA, USA). Pharmaceutical oral solution composed of sodium, potassium, and magnesium as sulfates (concentration 17.5 g/3.13 g/1.6 g, respectively, per 6 ounces) was purchased from the local Indian market manufactured by Lupin pharmaceuticals Pvt. Ltd. (Mumbai, India). Methane sulfonic acid was analytical grade and was procured also from Sigma-Aldrich (Burlington, MA, USA). All other chemicals and solvents used were of analytical grade. Water used in the HPLC analysis was prepared by Arium^®-611UFwater purifier from Sartorius (Göttingen, Germany).

2.2. Chromatographic Conditions

Chromatographic separations were executed on the DIONEX ICS-5000^® system connected to the Dionex^® suppressor (ASRS300 4 mm) and Dionex ICS-5000+^® conductivity detector from Thermo Scientific (Waltham, MA, USA). A guard column Dionex^® IonPac CG16^® (5 × 50 mm) was connected to the Dionex^® IonPac^® CS16 column (5 × 250 mm) column from Thermo Scientific (Waltham, MA, USA). The column temperature was set at 40 °C.

Mobile phase A was prepared by accurately measuring 6.7 mL of methane sulfonic acid into 1000 mL of Milli-Q-Water and the solution was sonicated for 5 min. Mobile phase B was Milli-Q-Water. A mobile phase gradient program was carried out as shown in

Table 1. Gradient elution program.

Time (min)	Mobile Phase-A	Mobile Phase-B
0.00	15	85
20.00	45	55
20.01	15	85
30.00	15	85

at the mobile phase flow rate set at 1.2 mL/min. Detection was performed by conductivity with cell temperature of 35 °C and current set at 158 mA. The standards and samples were injected in 20 µL injection volume and the total run time was 30 min.

All eluents were degassed and pressurized under high-purity nitrogen to prevent dissolution of carbon dioxide and subsequent production of carbonate. An aqueous solution containing 20 mM methane sulfonic acid was used for elution of cations. An aqueous solution containing 4 mM sodium hydroxide served as eluent for anions. Elution was carried out at a flow rate of 1.2 mL/min and 25 µL was injected for both anion and cation determinations. The concentrations of each cation and anion in the samples were calculated using a calibration curve that produced the relationship between the amount of analyte and the peak area. All analyses were carried out in duplicate.

2.3. Preparation of Standard Solutions

Standard stock solutions for sodium, potassium, and magnesium were prepared by accurate dilution by serial dilutions using Milli-Q water. Linearity standards were prepared from stock solutions by serial dilutions in Milli-Q water. Sodium linearity standards were prepared in concentrations 80.0, 120.0, 160.0, 200.0, and 240.0 ppm, while those for potassium were prepared at concentrations 20.0, 30.0, 40.0, 50.0, and 60.0 ppm. Magnesium standards were prepared at concentrations 4.5, 6.8, 9.0, 11.3, and 13.5 ppm.

For accuracy and precision testing, three quality control standards were prepared from the analytical grade of the analytes by spiking sodium, potassium, and magnesium sulfates into a placebo solution at low (QCL), intermediate (QCM), and high (QCH) concentrations within the aforementioned specified linearity ranges. These concentrations were prepared at 80.0/20.0/4.5, 160.0/40.0/9.0, and 240.0/60.0/13.5 ppm for sodium/potassium/magnesium, respectively.

3. Results

3.1. Choice of Chromatographic Conditions

Two columns were examined for the analysis of cations under study at diverse concentration ratios, namely, Dionex^® IonPac^® CS16 and Dionex^® IonPac^® CS15. The best choice in separation efficiency was the Dionex^® IonPac^® CS16 column.

For detection of eluted analytes, the conductivity detector is known to be the most common and useful detector in IC. Conductivity detection gives excellent sensitivity and even can enhance the selectivity, especially when coupled with chromatographic techniques with almost low background interferences. Therefore, detection was performed by conductometry with the conductivity cell temperature set at 35 °C and current set at 158 mA.

In IC, the mobile phase generally consists of an aqueous solution of a suitable salt or mixtures of salts. Occasionally, a small percentage of an organic modifier can be introduced, where its choice is best based on that in which most of the ionic compounds are dissolved better than in others. However, for greener chromatography and based on the green analytical chemistry concepts [16], it was better to exclude any toxic or hazardous organic modifiers. Therefore, initially 1% sulfuric acid was selected as mobile phase A with ultrapure water as mobile phase B with different flow and different column temperatures. However, the observed peak shapes were not satisfactory for magnesium and potassium. Further addition of 4% of methane sulfonic acid solution was used as a mobile phase A and ultrapure water as a mobile phase B at isocratic elution (50:50, v/v) with 1.0 mL flow rate at room temperature. In addition, there was no base line separation between the analytes’ peaks. Finally, increasing the flow rate to 1.2 mL and elution mode from isocratic into gradient programmed, the analytes’ peaks were resolved. The column temperature, however, was changed from room temperature into 40 °C, to avoid the peak fronting of magnesium.

3.2. Method Validation

The method was validated in accordance with ICH guidelines [17].

3.2.1. Optimization of Chromatographic Conditions

Ion chromatography is the most popular analytical method used for the determination of anions and cations in various sample matrices. Satisfactory separation depends mainly on the column, mobile phase, and flow rate. These three variables were screened during optimization of chromatographic conditions, which was carried out using mixed cations or anions standard solutions. The IonPac^® CS12A and IonPac^® AS12A analytical columns were used for cations and anions separation, respectively. The flow rate was set to 1.2 mL/min for both cations and anions optimization. The results demonstrated that the isocratic elution with 20 mM methane sulfonic acid solution enabled a satisfactory separation for Na⁺, K⁺, and Mg²⁺ within 15 min, while peak tailing and longer retention time occurred when 15 mM methane sulfonic acid solution in the mobile phase was used. Isocratic elutions with 4, 7, 10, 15, and 20 mM sodium hydroxide solution were employed for anions separation. The results indicated that a 4 mM mobile phase could improve resolution for Na⁺, K⁺, and Mg²⁺ within 15 min. The resolutions for acetate and lactate were not satisfactory when isocratic elution with other concentrations were used.

3.2.2. System Suitability

System suitability was performed to verify the performance of the analytical instrument. Blank and standard solutions were injected into the IC system and the chromatograms were recorded. System suitability was established by injecting a standard solution of Na/K/Mg at concentration 160.0/40.0/9.0 ppm (QCM) for six times and calculating the relative standard deviation (RSD) of analytes’ response. RSD results of the analytes under study were all less than 0.1%.

3.2.3. Specificity

Specificity is demonstrated by absence of background interferences of blank with main analytes. Blank, standard, and spiked sample solutions were injected. No interfering peaks were observed due to blank and placebo at the retention time of sodium, potassium, or magnesium peaks in standard solution (Figure 1). The data confirmed that the values meet the acceptance criteria, indicating the method’s specificity.

3.2.4. Linearity

The analytical method linearity shows its capability to obtain results that are well defined mathematically compared to the concentration of analytes in samples within a given range. Five linearity standard solutions were prepared for each analyte within concentration ranges of 80.0–240.0, 20.0–60.0, and 4.5–13.5 ppm for sodium, potassium, and magnesium, respectively. The response peak areas were calculated as a function of concentration and linearity graphs were constructed. Regression coefficients were calculated for all linearity lines and all were found to be acceptable (R² = 0.999). The linearity equations were Y = 109.37X + 43.09, Y = 71.07X − 20.66, and Y = 207.62X − 27.10 for sodium, potassium, and magnesium, respectively.

3.2.5. Accuracy

The accuracy of an analytical method conveys the propinquity of agreement between the true and found values. The accuracy was performed by injecting the QCL, QCM, and QCH standards and the assay of recovery results.

Table 2. Accuracy and precision results for determination of the analytes under study using the proposed method.

Standard	Sodium Sulfate		Potassium Sulfate		Magnesium Sulfate
	R% *	RSD **	R% *	RSD **	R% *	RSD **
	Accuracy
QCL	98.30	0.21	98.80	0.20	97.14	0.20
QCM	98.20	0.10	98.51	0.15	98.62	0.10
QCH	99.30	0.15	99.11	0.15	99.30	0.15
	Repeatability
QCM	100.80	0.42	99.82	0.41	100.37	0.41
	Intermediate Precision
QCM within 3 days	100.60	0.23	99.71	0.16	100.36	0.23
QCM different analyst	100.71	0.34	99.87	0.31	100.6	0.32

* Average Recovery% = calculated conc./Actual conc. × 100; ** RSD: Relative standard deviation.

shows the percentage recovery results obtained. The data confirmed that the values meet the acceptance criteria.

3.2.6. Precision

An analytical method’s precision is the degree of conformity among individual test results. Repeatability was investigated by injecting a homogenous sample of a single batch (QCM) and analyzing the relative standard deviation of results for six times. The RSD (Table 2) for the results indicates that the method is giving consistent results for a single batch. The intermediate precision was carried out to ensure that the analytical results remained unaffected by the change in analyst or days. Two different analysts performed the analysis of the QCM standard for three consecutive days and RSD results were obtained. The results of the intermediate precision study are addressed in Table 2. The RSD% data confirmed that the values meeting the acceptance criteria for the difference in analysis days were satisfactory and less than 2.0%. Hence, the method is considered rugged.

Intermediate precision was assessed from nine determinations (three determinations daily over three days) using the same equipment, but performed on three consecutive days using three separately prepared batches of eluents. Under intermediate precision conditions, retention times and integrated peak areas of all tested analytes were stable with 0.3–3.5 and 0.2–6.3% RSD, respectively. These values are slightly higher than what was found for repeatability. Method precision was also assessed by comparing the variations among six replicates determinations of the same batch of IC technical concentrate with the oral preparation value (%RSD). All the %RSD values of cations and anions determinations were less than the corresponding %RSD, indicating that the developed method is precise.

3.2.7. Robustness

The analytical method robustness study determines its capacity to remain unaffected by small, deliberate variations in the method’s parameters and provides an indication of its consistency during normal usages. Prepared standard and sample solutions were injected into the chromatographic system at different variable conditions. Robustness of the method was assessed by varying the instrumental conditions such as flow rate (±10%) and column temperature (±5 °C). The deliberate changes in the method have no significant changes in the % sssay of sodium, potassium, and magnesium. The robustness results are addressed in

Table 3. Robustness of the proposed methods for the determination of sodium, potassium, and magnesium sulfates.

Magnesium Sulfate *	Potassium Sulfate *	Sodium Sulfate *	Parameter
100.27 ± 0.59	99.83 ± 0.31	99.85 ± 0.50	Flow rate ± 0.12 mL/min
100.29 ± 0.39	100.1 ± 0.25	99.87 ± 0.68	Column temp. ± 5 °C

* Average %recovery ± %RSD results (n = 3).

. The data confirmed that the values meet the acceptance criteria. Hence, the method is robust.

3.3. Stability of the Analytical Solutions

The stability of the analytical solution is required for a reasonable time to generate reproducible and reliable results. The standard solution was prepared and performed under solution stability study at room temperature. Solution stability was evaluated after different intervals of 0, 21, 35, and 55 h at room temperature.

The results are addressed in

Table 4. Standard solution stability at room temperature.

Solution Stability (Hours)	Sodium		Potassium		Magnesium
	%Assay	%Difference	%Assay	%Difference	%Assay	%Difference
Initial	100.0	-	100.0	-	100.0	-
21 h	100.1	0.1	100.2	0.2	100.2	0.2
35 h	100.1	0.1	100.1	0.1	99.9	0.1
55 h	100.6	0.6	100.6	0.6	100.5	0.5

and data confirmed that the standard solution was found stable up to 55 h at room temperature condition.

3.4. Evaluation of the Proposed Method Compared to Reported Methods

The comparative evaluation of new analytical methodologies became an important step during the method development phase, which could help identify the pros and cons of each method, and hence improve its value. The proposed method was compared to previously published methods [13,18] for estimation of the same cations (

Table 5. Evaluation of the proposed method against reference methods [13,18].

	Proposed Green IC Method	Reported Method [13]	Reported Method [18]
Technique	Green IC coupled with conductivity	ICP-OES and ICP-MS	Flame AAS
Matrix	Pharmaceutical dosage forms	Human blood and serum	Food (Chocolate)
Working ranges
K (µg/mL)	20.0–60.0	20.0–40.0	1.0–10.0
Na (µg/mL)	80.0–240.0	20.0–40.0	1.0–3.0
Mg (µg/mL)	4.5–13.5	10.0–40.0	0.5–4.0
Sample preparation	None	Microwave digestion for 16 min	Sample emulsificaion using Tween 80 and Triton X100
Total analysis time	17 min	Average 20 min	Average 30 min
GAPI Greenness assessment

). The working ranges are shown, along with the matrices used for application. The proposed method is the most simple and most wide in range, which is suitable for a pharmaceutical product routine evaluation without need for complex sample preparation steps, such as microwave digestion or surfactant extraction.

Other main evaluation criteria are the ecological impact of the method and sustainability of the procedures [19,20]. Several metrics have been developed recently for the assessment of greenness as part of green analytical chemistry (GAC) concepts. One of the first developed metrics for assessing the GAC applied concepts was the national environmental methods index (NEMI) [21]. NEMI is a symbolized figure of a circle divided into only four sections. The sections describe the reagents used in an analytical technique in the form of their corrosive, health hazards, PBT hazards (persistent, bio-accumulative, and toxic), and generated waste. NEMI was the seed research that encouraged other scientists to develop other metrics covering the shortage of previous ones. The analytical eco-scale was then introduced in 2012 by Galuszka et al. [22]. The analytical eco-scale did not use a pictogram; however, it was the first scoring for assessment. Penalty points were counted for each factor within the analytical procedure that did not follow the GAC concepts and those penalty points were deducted from a score of 100. Among the developed metrics, the green analytical procedure index (GAPI) has been markedly cited and utilized [23]. GAPI is a color-lead pictogram comprising five pentagrams representing fifteen steps of the analytical method starting from the sampling, going through instrumentation, reagents, and generated waste [24]. The red color indicates a high ecological impact, while yellow or green ones are better environmental sustainability. Then, another Hexagon metric was introduced in 2019 [25], however, it did not cover the whole steps of the analytical methodology as in GAPI. In 2020, the AGREE metric was published together with simple software that was easy to use and reproduce [26]. AGREE considered both the numerical evaluation and the symbolic one. Although AGREE did not consider each step within the analytical method as in GAPI, AGREE covered the whole 12 concepts of GAC carefully. Both AGREE and GAPI are currently the highest in use and citation by analytical chemists and researchers [19]. The latest metric was introduced in 2021 and was called the RGB-12 algorithm [27]. The RGB-12 algorithm added new socio-economic dimensions to the assessment process through inventing 12 new white chemistry principles as an alternative to GAC. However, this metric still needs more improvements.

The proposed method was evaluated against GAPI to assess every step within the analytical procedure, as shown in Table 5. Most of the steps are green according to GAC concepts. Only red colors of the steps of the off-line sampling are needed for transfer of such samples between the obligatory segregation of the pharmaceutical production from the quality control laboratories, which is also the same conditions for the other two methods under comparison. Another red zone is located in the amount of waste generated, as shown in the lower right pentagram. However, the collective evaluation of this pentagram, which represents instrumentation, can be estimated as almost green since the energy utilization and type of waste generated are moderate to green. Ieggli et al. [18] published an AAS technique for estimation of the cations under study in emulsified chocolate. As shown in their GAPI assessment (Table 5), the instrumentation has better energy consumption. However, the sample preparation steps are tedious for their established matrix, besides the use of ecologically friendly surfactants, still the amount needed is much higher compared to the proposed method utilization of chemicals. The worst scenario in energy utilization was with the published ICP-OES/MS method [13], with a long time and high energy digestion (Energy >1.5 KWH) and use of strong acids for sample digestion.

The ecological aspects of ion chromatography were recently reported [28]. Michalski and Pecyna-Utylska concluded that IC is currently a dominating technique when it comes to the routine analysis of inorganic ions. As major aims of GAC encompass the low cost, low environmental danger, low energy consumption, and safe eluents [29], IC is miniaturized and doubtfully green [28]. Moreover, it generates lower waste and could be moved into the location of sampling for in situ analysis, and thus saves the analysis and transportation cost. The most important limitation for IC considering greenness is its labor intensive property when it comes to sample preparation steps for solid samples [30].

4. Conclusions

Currently, the high demand for sensitive and selective tools for a routine quality control of pharmaceutical products must be compromised with their ecological impact. Ion chromatography is a well-established tool, that when coupled with conductometric detection, their combined selectivity is augmented. As ion chromatography can selectively separate different cations, also conductometry can differentiate the oxidation states of the same cation, and hence their selectivity is augmented. Meanwhile, the estimation of inorganic cations in pharmaceutical dosage forms remains as one of the most important applications of ion chromatography. The proposed newly developed sensitive and selective ion chromatographic method was validated for the simultaneous indirect determination of sulfate salts as sodium, potassium, and magnesium. The method was found to be highly specific and has the advantages of being organic-solvent free and of greener ecological impact. The method can be used in routine analysis of the analytes’ oral solution and stability testing as a good alternative to ICP-OES/MS procedures with higher cost-effectiveness. Therefore, analytical methods based on ion chromatography should become more integrated in regulated pharmacopoeial monographs at higher selectivity levels and lower limits for the upcoming future.