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Determination of Inorganic Ions in Parenteral Nutrition Solutions by Ion Chromatography

https://doi.org/10.3390/molecules27165266

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.

2.1.1. IC Separation Issues

As starting conditions, a cation analysis method for column testing was applied: cations were separated with an IonPac CS16 column kept at 40 °C and the mobile phase was 30 mM MSA with a flow rate of 0.36 mL/min [18]. The PN sample was injected following simple dilution with Milli-Q water. The chromatogram showed that the amino acids interfered with the determination of sodium and calcium (Figure 1). Therefore, adjustment of the chromatographic parameters was explored, in an attempt to separate peaks of analytes from interfering components. Different mobile phases were used, including 20 mM MSA, 30 mM MSA, 40 mM MSA, 30 mM MSA with 5% acetonitrile (ACN), 30 mM MSA with 10% ACN, 2 mM MSA with 10% ACN and 30 mM MSA with 5% tetrahydrofuran. A lower concentration of MSA or organic modifier could obviously delay the retention times, but this had limited improvement on the separation of sodium and the interfering substances. In this way, 30 mM MSA was maintained for further experiments. When changing the temperature in steps of 2 °C from 40 °C to 34 °C, peaks shifted only slightly. The best selectivity was obtained at 36 °C, but sodium and calcium ions could not yet be properly determined. In a next step, cleanup of the sample was explored.
Figure 1. Chromatogram of blank, standard solution of cations in the PN solution, mixture of amino acids and PN solution without pretreatment. Peak 1: sodium, peak 2: potassium, peak 3: magnesium and peak 4: calcium.

2.1.2. Cleanup Procedure

Solid-phase extraction (SPE) has been reported to extract amino acids [19,20]. Three different SPE cartridges of HLB and MCX (Waters, Milford, MA, USA) and OnGuard II A (Thermo Scientific, Sunnyvale, CA, USA) were applied to extract the amino acids in this research. The experimental procedure was based on the operation manual and methods reported in literature [20,21]. For HLB, the cartridge was rinsed with 2 mL of methanol (MeOH) followed by 2 mL of Milli-Q water. Then, the sample that was acidified with 0.2% formic acid (FA) was loaded. Next, 2 mL of MeOH containing 10% Milli-Q water and 0.1% FA was used as washing solution. Subsequently, 500 µL of collected solution was dried and re-dissolved in 1 mL of 30 mM MSA for further analysis. For MCX, the cartridge was rinsed with 2 mL of MeOH followed by 2 mL of 1% FA. The next steps (loading, washing and re-dissolving) were the same as for the HLB cartridge. For OnGuard II A, the cartridge was conditioned with 10 mL of 30 mM MSA, followed by 10 mL of Milli-Q water. Loading 5 mL of sample solution, the first 3 mL of effluent were discarded, and the next 2 mL were collected for analysis. Although some cleanup was realized (see Figure 2), there was still interference, especially with the determination of sodium. Since there are over twenty amino acids in the PN solution, it is hard to totally remove the amino acids from the sample and so attempts using SPE were found to be unsatisfying.
Figure 2. Chromatogram of sample solution pretreated by MCX, HLB and OnGuard II A cartridge. Peak 1: sodium, peak 2: potassium, peak 3: magnesium and peak 4: calcium.
Another approach is to heat the PN solution at a high temperature to decompose the amino acids, while metal ions such as sodium, potassium, magnesium and calcium remain in the residue. According to the European Pharmacopoeia (Ph. Eur.), methods for total ash and sulfated ash are able to remove amino acids from samples [22]. Therefore, the applicability of both methods as such was examined. After calcination, the residues were dissolved in water and injected in the IC system. Both methods provided an efficient cleanup as no further interference with the peaks of interest in the chromatogram was noticed. However, the recovery (Table 1) for Ca2+ and Mg2+ was below 10% for both procedures. Changing from water to 30 mM MSA as pickup solvent yielded recovery values for Ca2+ and Mg2+ above 90% (Table 1) with a slightly better performance for the sulfated ash method, while the recovery values for Na+ and K+ were more than 10% too high. As the H2SO4 used in this procedure could be a source of Na+ and K+, concentrated HNO3 and MSA, as well as 1 M MSA were also considered as calcination media. The results are presented in Table 1. Finally, 1 M MSA was withheld as the calcination medium with 30 mM MSA as the pickup solution.

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