1. Introduction
Parenteral nutrition (PN) is administered intravenously and has been applied in clinical practice for over 50 years [
1]. Currently, PN is used in several therapies of patients with short bowel syndrome, gastrointestinal bleeding, bowel obstruction and absorption disorders [
2]. It also provides nutrition support for cancer patients, geriatric populations and preterm infants who have poor nutrition intake [
3,
4,
5]. As PN is mainly used for feeble patients, the compounding of PN solutions in hospital pharmacies needs a strict control for quality assurance [
6]. PN solutions usually contain over 20 ingredients, including electrolytes, glucose, amino acids and trace elements, and these nutritional solutions are typically prepared in hospital pharmacies. To avoid the risk for patients caused by errors in the electrolyte concentrations, quality control should be performed but is often limited [
6]. Currently, sodium, potassium, magnesium and calcium are usually analyzed by potentiometric or photometric/spectrometric methods [
7]. However, reproducibility of these methods is rather poor, they can be labor intensive, and the large amount of amino acids in PN solutions may interfere with the determination of the ions [
7,
8]. Therefore, other analytical techniques are required.
There are some widely used techniques for analyzing inorganic ions, such as atomic absorption spectroscopy (AAS), capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C
4D), inductively coupled plasma quadrupole mass spectrometry (ICP-MS) and ion chromatography (IC) [
7,
9,
10,
11,
12,
13,
14]. Some of them, such as CE-C
4D and ICP-MS have been developed to analyze inorganic ions in PN solutions [
7,
14]. The drawbacks of CE are poor reproducibility of migration times and peak areas, which restrict its use for routine analysis [
15]. The widespread use of ICP-MS is limited due to its high cost.
IC is a variant of high-performance liquid chromatography. As a highly sensitive and selective technique for the determination of inorganic ions, it has been used for routine determination of cations and anions in a wide variety of samples in many industries, including environment, biotechnology, agriculture, food and pharmaceutical industries [
16,
17]. Numerous methods were developed to determine sodium, potassium, magnesium, calcium and chloride utilizing IC with as main benefits compared to other analytical techniques: short analysis time, excellent sensitivity, high selectivity, small sample volume and cost-effectiveness [
8,
15]. Cation-exchange columns and anion-exchange columns have been applied for the determination of the respective ions due to their affinity to the specific ion exchanger. Conductivity detection is a universal detection technique in IC, including the suppressed and non-suppressed mode. For the suppressed mode, a suppressor is installed after the analytical column to lower the background signal and increase the sensitivity. For the non-suppressed mode, there is only a conductivity detector after the column making the system simpler and cheaper. Even though IC is commonly used in inorganic ions analysis, no study has been found in the literature describing the determination of sodium, potassium, magnesium, calcium and chloride in PN solutions.
In this study, IC methods for the determination of sodium, potassium, magnesium, calcium and chloride in PN solutions were developed using a one-factor-at-a-time approach. This was preferred above a design of experiments because influencing factors (including non-continuous ones) were difficult to predict in advance. Mainly, issues related to the sample pretreatment were encountered and solved step by step. After optimization, the methods were validated.
3. Materials and Methods
3.1. Reagents and Materials
Standards are prepared from sodium chloride (99.87%, Merck KGaA, Darmstadt, Germany), potassium chloride (99.5+%, Chem-lab, Zedelgem, Belgium), magnesium chloride (98+%, Sigma-Aldrich, St. Louis, MO, USA) and calcium chloride (97+%, Sigma-Aldrich). MSA (>99.0%, Sigma-Aldrich) and sodium hydroxide (50% w/w aqueous solution, Acros Organics, Geel, Belgium) were purchased for mobile phase preparation. Water was purified by a Milli-Q water purification system from Millipore (Bedford, MA, USA). The oven (MR 170) was purchased from Heraeus (Hanau, Germany).
Glassware should be avoided for the preparation and storage of solutions and replaced by synthetic material.
3.2. Preparation of Standards and Samples
3.2.1. Preparation of Standards
MSA was diluted to 1 M and stored in a plastic bottle at 4 °C as a stock solution. The mobile phase for cation determination was prepared by diluting 1 M MSA to 30 mM MSA in Milli-Q water. Sodium hydroxide (NaOH) was diluted to 20 mM as the mobile phase for anion determination.
Standard stock solutions of the analytes were prepared by dissolving NaCl, KCl, MgCl2 and CaCl2 in Milli-Q water to obtain concentrations of 22.05 mM for Na+, 14.05 mM for K+, 1.75 mM for Mg2+, 10.50 mM for Ca2+ and 4.57 mM for Cl− and stored in plastic bottles at 4 °C for no more than one week. Stock solutions were diluted to the standard solutions ranging from 50 to 150% of the test solution for the linearity tests. Stock solutions of Na+, K+, Mg2+ and Ca2+ were mixed and diluted 10 times as 100% standard solution for cation determination. For anion determination, the Cl− stock solution was diluted 2 times as 100% solution.
3.2.2. Preparation and Quantification of Inorganic Ions in PN Solution
The PN solutions were prepared in the hospital pharmacy of UZ Leuven (Leuven, Belgium). The composition of all components is shown in
Table 9. A calcination method was developed as a sample pretreatment method for cation analysis: 1.0 mL of PN solution was pipetted in a quartz crucible, then 1 mL of 1 M MSA was added to provide an acidic environment to facilitate calcination. After digesting at 100 °C for 1 h, the residue was heated with a Bunsen burner until white fumes were no longer evolved. Next, the crucible was heated at 550 °C for 1 h and cooled in a desiccator. The residue was dissolved in 20 mL of 30 mM MSA and filtered through a 0.20 µm filter as a test solution for injection. To determine Cl
− in the PN solution, the latter was diluted 20 times with Milli-Q water and filtered for injection.
The concentrations of the four cations and the anion in the test solutions were: 50.72, 54.94, 4.20, 41.3 and 81.12 mg/L for Na+, K+, Mg2+, Ca2+ and Cl−, respectively. All solutions were prepared in duplicate, and each was injected in triplicate.
3.3. Instrumentation and Chromatography Conditions
Analyses were conducted using an IC system (ICS-3000, Dionex, Sunnyvale, CA, USA), equipped with a DP analytical pump, an AS50 auto-sampler, a conductivity detector (CD), a Dionex CSRS 300 suppressor for cation determination and a Dionex ADRS 600 suppressor for anion determination. Chromeleon 6.8 was used for data collection and system control.
An IonPac CS16 analytical column (3 mm × 250 mm) from Thermo Scientific (Sunnyvale, CA, USA) was used for cation separation. The eluent was 30 mM MSA at a flow rate of 0.36 mL/min and the column temperature was 36 °C. An IonPac AS19 analytical column (4 mm × 250 mm) from Thermo Scientific was used for anion separation. The eluent was 20 mM NaOH with a flow rate of 1.0 mL/min and the column temperature was 35 °C.
3.4. Validation Test
The method was validated for selectivity, sensitivity, linearity, precision, recovery and robustness according to the ICH guidelines [
24].
3.4.1. Selectivity
Selectivity was examined by analyzing the standard solution and blank solvent (Milli-Q water) to ensure the separation of the different ions and possible interference from the blank solution consisting of Milli-Q water.
3.4.2. Sensitivity
In order to check the sensitivity of the method under the working conditions used, the LOD and LOQ were determined at a signal-to-noise ratio of 3 and 10, respectively.
3.4.3. Linearity
Calibration curves of different ions were prepared in five concentrations of standard solution ranging from 50 to 150% of the test concentration: 25.36 to 76.07 mg/L for sodium, 27.47 to 82.40 mg/L for potassium, 2.10 to 6.29 mg/L for magnesium, 20.65 to 61.95 mg/L for calcium and 40.56 to 121.68 mg/L for chloride.
3.4.4. Precision
Precision of the IC methods was evaluated by the repeatability and intermediate (inter-day) precision. Repeatability was determined by 6 replicates at 3 levels (80%, 100% and 120% of the test concentrations of the ions) on day 1, while the inter-day precision was evaluated by injecting the 100% solution in triplicate on days 2 and 3.
3.4.5. Recovery
The PN solution is a drug product of which the contents of the ingredients are known. The ingredients were separately available. The recovery of the ions was determined by adding three different concentration levels (80%, 100% and 120% of test concentration) of standard solution to a mixture of components in the PN solution which were not subject of the analysis. The sample pretreatment method was applied for the cations. The recovery was calculated using the following equation:
3.4.6. Robustness
A robustness test was performed applying an experimental design to ensure the reliability of the analytical method by slightly varying the chromatographic factors within a certain range. In this study, a two-level full factorial design was applied. Three chromatographic parameters (suppressor current, concentration of MSA in the mobile phase and column temperature) were investigated at two levels (−1 and +1) around their central level (0) (
Table 10).
According to the experimental design, 2
k experiments were performed in random order, where
k is the number of factors. Another three experiments with the variables at the central level were carried out at the beginning, middle and end of the series. In total, 11 experiments were carried out in this test. The mathematical relationship between the experimental variables (
xi,
xj, …) and response (
y) can be obtained from the following equation:
where
b are the regression coefficients and
ε is the experimental error.
b0 stands for the intercept,
bi and
bj describe the quantitative effect of the respective variables
xi and
xj, and
bij represents the interaction effect between both variables. As responses, the resolutions Rs
Na-K, Rs
K-Mg and Rs
Mg-Ca as well as the peak areas of Na
+, K
+, Mg
2+, Ca
2+ and Cl
− were selected.