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Minimizing LCMS Artifacts Using Arium® Ultrapure Water Systems
Liquid Chromatography-Mass Spectrometry (LCMS) is a sophisticated analytical technique that seamlessly integrates the separation capabilities of liquid chromatography with the precise identification and quantification abilities of mass spectrometry. LCMS has become indispensable in modern analytical chemistry, f inding extensive application across diverse scientific domains such as pharmaceuticals, environmental analysis, food safety, metabolomics, proteomics, and forensic science.
In achieving analytical excellence, ultrapure water plays a pivotal role in LCMS applications. Ultrapure water, conforming to ASTM Type I specifications, underpins the entire analytical workflow, ensuring precision, reliability, and accuracy in results. Mitigating the challenges posed by water impurities, including organic compounds, bacteria, and ions, is essential for achieving optimal performance in both chromatography and mass spectrometry. By employing advanced water purification systems like the Arium® Mini UV, scientists and laboratory technicians can access ultrapure water of exceptional quality, thereby enhancing the sensitivity and reliability of analytical outcomes.
This article delves into the critical importance of ultrapure water in LCMS, examines the impact of water impurities on chromatographic performance, and highlights the advantages of using state-of-the-art water purification systems to ensure consistent, reliable, and precise analytical results.
Effects of Impurities
Organic Compounds
Organic compounds in the mobile phase are among the most significant contaminants in liquid chromatography applications. When present, these compounds compete with analytes for binding sites on the stationary phase. This competition diminishes analyte retention on the column, thereby reducing method sensitivity.
Over time, organic compounds can accumulate on the column surface, obstructing analyte and solvent access to active sites and leading to mass transfer inefficiencies and a loss of resolution. If such contaminants concentrate at the column's head, they may cause ghost peaks, interfering with the separation process. In severecontamination scenarios, organic compounds may form a pseudo-stationary phase, resulting in peak tailing, retention time shifts, and compromised analytical accuracy.
Bacterial Contamination
Bacteria and residual biofilms in water can obstruct columns and frits, causing flow irregularities and degraded chromatographic performance. Organic by-products produced by bacteria, such as pyrogens, nucleases, or alkaline phosphatase, can induce chromatographic disturbances, adversely affecting the accuracy, precision, and reproducibility of results. These contaminants also compromise the stability and longevity of the chromatographic column.
| Conductivity [µSxcm-1] | Resistivity [MΩ x cm] | TOC [ppb] | Endotoxin [EU x ml-1] | Bacteria [CFC x mL-1 | |
| Arium® Mini UV | 0.055 | 18.2 | ≤5 | 0.001 | 0.001 |
Inorganic Contamination
Excessive alkali ions and other inorganic impurities can introduce noise and skew results in LCMS analysis. To mitigate these risks, it is recommended to replace mobile phases rather than topping them off, thereby preventing the accumulation of contaminants in supply bottles. Prolonged exposure of mobile phases to air can also lead to phthalate contamination, underscoring the necessity for a clean and controlled environment.
Ionic impurities in water can alter the ionic strength of a solution, impacting specific chromatographic separations. Changes in ionic strength influence conductivity, osmotic pressure, and the behavior of charged species, thereby affecting overall chromatographic performance.
Advantages of Water Purification Systems
Commercially available LCMS-grade bottled water may yield suboptimal results due to contamination during production or inadequate bottle handling. For sensitive LCMS applications, the use of freshly produced, high-quality ultrapure water is crucial to ensuring accurate and reliable outcomes.
Sartorius’ Arium® Mini UV systems deliver ultrapure water meeting ASTM Type I standards with attributes such as high resistivity, low Total Organic Carbon (TOC) levels, and minimal bacterial content. These systems ensure the highest water purity, enabling reliable and accurate chromatographic data.
Total Organic Carbon (TOC)
TOC serves as a practical indicator of organic contamination in water. This method oxidizes organic substances and measures the resultant products to determine contamination levels.
Ultraviolet Radiation
UV radiation is a key mechanism in breaking down and oxidizing organic compounds. A wavelength of 185 nm effectively disintegrates carbon-containing molecules, producing ionized fragments removable by ion exchange. Additionally, UV light inactivates microorganisms by damaging their RNA and DNA, ensuring water purity.
Arium® Mini UV systems utilize full-spectrum UV lamps to maximize organic molecule breakdown, ensuring water quality suitable for high-performance liquid chromatography applications.
Arium® Mini UV Ultrapure Water Systems
Materials and Methods
Feasibility studies conducted at Sartorius Bohemia, NY, USA, evaluated the impact of various water sources on LCMS performance. A DG44 CHO-cell mAb production process was carried out using the Sartorius Ambr® 15 platform, followed by analysis with the Waters BioAccord™ LCMS System. Samples were processed using Arium® ultrapure water, commercially available LCMS-grade bottled water, and in-house utilities deionized (DI) water.
The findings revealed that both Arium® ultrapure water and LCMS-grade bottled water displayed comparable chromatographic profiles with consistent peak shapes and baseline stability. In contrast, in-house DI water exhibited elevated backgroundlevels and lower peak intensities, indicating the presence of impurities. Mass spectral analysis further identified specific contaminants in DI and bottled water samples, reinforcing the need for ultrapure water for LCMS applications.
Arium® Mini UV Ultrapure Water Systems
Compact and Reliable
The Arium®MiniUVsystemisdesigned to meettheultrapure water requirements of smaller laboratories, offering a daily output of 10 liters from either tap or pre-treated water. Its compact design (28 cm width) makes it suitable for labs with space constraints. Equipped with user-friendly displays and innovative bag-tank technology, the system ensures reliable results for routine and critical analyses alike.
Arium® Mini UV Ultrapure Water Systems
| Water Condotions | Water Source | Stated Resistivity |
| 1 | Arium® Mini UV | >18 MΩ |
| 2 | Utilities USP Water | >18 MΩ |
| 3 | Commercial LCMS Bottled Water | Not Stated |
| ResultsLC Conditions | |
| ResultsLC System | Water ACQUITYTM Premier System |
| Detection | TUV |
| Column | 2.1 x 100 mm BioResolveTM RP Polyphenyl |
| Column Temp | 60 0C |
| Sample Temp | 8 0C |
| Injection Volume | 5µL |
| Flow Rate | 0.4mL/min |
| Mobile Phase A | H2O, 0.1% Formic Acid (Range of Conditions) |
| Mobile Phase B | Acetonitrile, 0.1% Formic acid |
| MS Conditions | |
| MS System | Water ACQUITY RDaTM |
| lonization Mode | Positive |
| Acquisition Range | 500 - 7,000 m/z |
| Capillary Voltage | 1.5 kV |
| Collision Energy | NA |
| Cone Viltage | 95 V |
Results
The water sources used in the analysis included freshly opened LCMS-grade bottled water, known for its high purity, and Arium®-water, which adheres to strict quality standards. The performance of in-house deionized (DI) water (USP), a common and cost-effective water source in laboratory settings, was also evaluated.
Figure 1. Total Ion Chromatogram (TIC; 1; TOF MS (400 – 7000), 95 V ESI+), Mass Spectral Data (2; Average Time 2.1751 min, TOF MS (400 – 7000), 95 V ESI+: Combined) From a Monoclonal Antibody Sample And UV Traces (3; Channel Name: TUV 280)
The data analysis revealed some notable trends. Both LCMS-grade bottled water and Arium®-water showed similar chromatographic profiles, with comparable peak shapes and stable baselines, indicating that these high-purity water sources perform consistently and effectively in LCMS analysis. However, when in-house DI water (USP)was used, the chromatographic profiles showed a significantly higher background level, suggesting the presence of impurities or contaminants such as TOC, inorganic substances, and bacterial contamination. Additionally, a slight variation in peak area was observed, highlighting the potential impact of water quality on chromatographic accuracy and reliability.
Figure 2.Extracted Mass Chromatogram of Retention Time Time ~1.6–1.7 Min (Light Chain Peak, 1) And Zoomed in Spectra (2); Average Time 2.1168 min, TOF MS (400 – 7000), 95 V ESI+: Combined.
The peak at around 1.61 minutes corresponds to free Light Chain in the sample and was visible across all water conditions. This is typical for unpurified cell-culture samples, as the Protein A purification step was excluded, allowing other proteins to remain in the solution. The larger peak at around 2.08 minutes corresponds to the complete monoclonal antibody (mAb), which was clearly differentiated in all water conditions. While Arium®-water and bottled water showed similar curves, the USP water resulted in a lower peak intensity and higher background absorbance.
Figure 3. Total Ion Chromatogram(TIC; 1, TOF MS (400 – 7000), 95 V ESI+) And Mass Spectrum From a Monoclonal Antibody Sample Prepared With Arium®-water Arium®-water (Average Time 2.1418 min (UV3) and 2.1668 min (PERF), 2, TOF MS (400 – 7000), 95 V ESI+: Combined).
Further analysis of the mass spectra of the free light chain in solution revealed a potential contaminant in the lower m/z range of some samples. Upon closer inspection, this contaminant was only present in the USP and bottled water mobile phase samples. The isotopic abundances of the ion were consistent with a molecular formula of C24H19N6O2Cl2. Although the exact structure was not determined, the difference in abundance was clearly noticeable.
Due to its proven performance and reliability, Arium®-water was fully implemented in the Sartorius upstream laboratory for LCMS applications. Figure 3 shows the intact mass results from a perfusion CHO cell culture, with samples taken both from inside the bioreactor and from the perfusate path (PERF).
The data reveals small differences in relative glycoform abundance between the two samples, with minimal background noise, making the results easy to interpret.
Figure 4. Comparison of Expenses Between In-House Produced Arium®-water Type I (Ultrapure Water) and Bottled Water(LCSM grade With PFAS Content Below Detection Limit)
Conclusion
This study underscores the critical role of water quality in LCMS applications. Inadequate water sources, such as in-house DI water or improperly handled bottled LCMS-grade water, can introduce contaminants that compromise the accuracy and reliability of analytical results. The use of advanced water purification systems, such as the Arium® Mini UV, ensures ultra-pure water that meets stringent quality standards, enhancing chromatographic performance and laboratory efficiency.
nvesting in in-house water purification systems is not only scientifically advantageous but also economically beneficial. Over a year, laboratories using Arium® systems can achieve significant cost savings while ensuring superior data quality. By prioritizing ultrapure water, laboratories can achieve precise, reliable, and reproducible results, cementing the foundation for advanced analytical research and applications.
References
1. Williams, S. Ghost peaks in reversed-phase gradient HPLC: a review and update. Journal of chromatography. A 1052, 1–11; 10.1016/j.chroma.2004.07.110 (2004).
2. Bendlin, H. & Eßmann, M. Reinstwasser. Planung, Realisierung, Qualifizierung von Reinstwassersystemen. 1st ed. (Maas & Peither GMP-Verl., Schopfheim, 2004).
3. Choi, J. & Chung, J. Effect of dissolved oxygen on efficiency of TOC reduction by UV at 185 nm in an ultrapure water production system. Water research 154, 21–27; 10.1016/j.watres.2019.01.037 (2019).
4. Pereira, R. V. et al. Evaluation of the effects of ultraviolet light on bacterial contaminants inoculated into whole milk and colostrum, and on colostrum immunoglobulin G. Journal of dairy science 97, 2866–2875; 10.3168/ jds.2013-7601 (2014).