Identification and Quantitation of NNitrosamines in Normal Saline Intravenous Infusion bags Using UHPLC-MS/MS

Identification and Quantitation of N‑Nitrosamines in Normal Saline Intravenous Infusion Bags Using UHPLC‑MS/MS — Summary

Significance of the topic

N‑Nitrosamines are potent mutagenic/carcinogenic impurities that have become a major pharmaceutical safety concern since 2018. Their detection in finished drug products and potential formation from packaging or manufacturing excipients has prompted evolving regulatory expectations and the need for highly sensitive, reliable analytical methods. Intravenous (IV) infusion bags present a particular focus because leachable nitrosamines (e.g., NDBA, NDMA, NDEA) can contaminate sterile parenteral solutions, and in some clinical scenarios, exposure may be prolonged. Analytical approaches able to detect low pg/mL levels, demonstrate selectivity in aqueous saline matrices, and support regulatory-quality workflows are therefore essential for risk assessment and control.

Objectives and overview of the study

This application note describes development and demonstration of a targeted UHPLC‑MS/MS method for identification and quantitation of seven small dialkyl N‑nitrosamines in 0.9% NaCl IV infusion bags. Goals included achieving very low limits of detection/quantitation (pg/mL level), resolving isobaric compounds, mitigating system- and solvent-derived interferences, and establishing a compliant-ready data acquisition and review workflow.

Methodology

Key elements of the analytical approach:

• Sample handling: IV bag contents were transferred directly to autosampler vials; matrix calibration standards and QCs prepared in nitrosamine‑free 0.9% NaCl.
• Chromatography: ACQUITY Premier HSS T3 column (2.1 x 100 mm, 1.8 μm) at 45 °C with ammonium formate/formic acid buffers; a delay column (Atlantis Premier BEH C18 AX) was placed upstream to separate persistent system/mobile-phase background that coelutes with NDBA. The first 1.75 min of LC flow was diverted to waste to avoid transmitting saline to the MS source.
• Mass spectrometry: Xevo TQ Absolute XR triple quadrupole operated in positive APCI with optimized MRM transitions. Soft transmission settings were applied for some analytes to reduce in‑source fragmentation. Targeted MS/MS experiments and QTOF exact-mass confirmation were used to increase identification confidence for selected findings.
• Data processing and QA/QC: waters_connect for Quantitation coordinated MRM optimization, acquisition, automated calibration/QC rule‑sets, blank checks, ion‑ratio flagging, and reporting. A weighted 1/x linear regression was used without internal standards.

Used instrumentation

• UHPLC: ACQUITY Premier UPLC System with Binary Solvent Manager and FTN sample manager.
• Analytical column: ACQUITY Premier HSS T3, 1.8 µm, 2.1 x 100 mm.
• Delay column: Atlantis Premier BEH C18 AX, 2.1 x 50 mm (used to mitigate persistent interference for NDBA).
• MS (quantitation): Xevo TQ Absolute XR triple quadrupole, APCI+ ionization, MRM acquisition.
• MS (confirmation): Xevo G3 QTOF high-resolution MS for elemental composition confirmation.
• Detector/accessories: 2998 PDA for UV monitoring; waters_connect for acquisition, processing and 21 CFR Part 11‑ready data management.

Main results and discussion

Analytical performance:

• Linear calibration for seven nitrosamines across 0.01 (or 0.02)–100 ng/mL with R2 > 0.99 using 1/x weighting, providing approximately four orders of linear dynamic range.
• LLOQs in the low pg/mL range (lowest calibration points tested down to ~0.005–0.01 ng/mL with acceptable blank levels and signal‑to‑noise).
• QC performance (matrix QCs at 0.075, 0.75, 7.5, 75 ng/mL, n=6): mean accuracies 88–110% and %CVs 0.033–5.5% across analytes and levels.
• Matrix effects measured across several concentrations showed modest suppression/enhancement between −9.3% and +13.2%, with SDs up to ~11%—overall minimal impact on quantitation when using matrix calibration.
• No internal standards were employed; method acceptance relied on matrix calibration, QC levels and software rule‑sets.

Interference management and identification confidence:

• System or solvent‑derived interference coeluting with NDBA was mitigated by inclusion of a delay column and by diverting early flow to waste; this improved selectivity and lowered detection limits.
• MRM ion‑ratio checking (±15% acceptance window) and targeted MS/MS spectra were used to distinguish true nitrosamine peaks from closely eluting or isobaric interferences. Example: NDEA detection in a 100 mL IV bag was supported by matching retention time, consistent MRM ion ratios and confirmatory MS/MS spectra; a second nearby peak exhibited different ion ratios and fragment patterns.
• QTOF exact mass further confirmed elemental composition for selected positive identifications (e.g., NDEA at the observed tR).

Application to commercial samples:

• IV bags purchased from an online vendor (Sample Set 1) yielded detections of NDMA and NDEA in some 100 mL bags; spike recovery experiments (0.1 and 1.0 ng/mL) returned 89.4–98.4% recovery with CVs 3.3–6.0%, supporting method accuracy in these matrices.
• A batch of 500 mL IV bags obtained with authorization from a pharmacy supplier (Sample Set 2, n=6) tested negative for all seven nitrosamines using the method.
• The toxicological significance relative to regulatory Acceptable Intake (AI) values remains context dependent; CDER has signalled investigation into NDBA and potential less‑than‑lifetime adjustment factors (approx. 4–10×) for infusion bag leachables.

Benefits and practical applications

• High sensitivity: low pg/mL detection enabling trace‑level monitoring of nitrosamines in aqueous IV matrices.
• Chromatographic resolution: separation of isobaric species (e.g., NDPA vs. NDIPA) using the HSS T3 phase and optimized gradient/delay column strategy.
• Robust identification: combination of retention time, MRM ion‑ratio flagging and targeted MS/MS (with optional HRMS confirmation) increases confidence for regulatory‑relevant identifications.
• Practical workflow: waters_connect supports method optimization, automated rule‑based data review, and 21 CFR Part 11‑compatible recordkeeping to facilitate routine testing and reporting.

Future trends and opportunities for use

Recommended directions and likely developments:

• Broader monitoring programs for nitrosamines and other leachables across different IV packaging materials, with expanded sample sizes and supplier traceability to establish prevalence and root causes.
• Standardization of less‑than‑lifetime adjustment approaches and harmonized regulatory guidance for leachable nitrosamines in parenteral products.
• Increased adoption of orthogonal confirmation strategies combining MRM ion ratios, targeted MS/MS spectra and high‑resolution exact mass to strengthen identifications in regulatory submissions.
• Implementation of isotopically labeled internal standards or surrogate standards to improve accuracy, correct for variability and increase confidence in routine quantitative testing.
• Continued use of automated, rules‑driven informatics platforms to accelerate result review, ensure data integrity, and simplify compliance with regulatory requirements.

Conclusion

The described UHPLC‑APCI‑MS/MS method achieves low pg/mL quantitation of seven small dialkyl nitrosamines in 0.9% NaCl IV infusion bags, with strong linearity, acceptable accuracy/precision, and manageable matrix effects. Strategic use of a delay column and flow diversion reduced system‑related interferences (notably for NDBA). Combining MRM ion ratios, targeted MS/MS and optional high‑resolution mass confirmation yields robust identification confidence suitable for investigatory testing and routine monitoring. The workflow, integrated with waters_connect software, supports compliant data handling and rapid review, but laboratories should perform full method validation and consider internal standardization before deployment in regulated testing programs.

References

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