(W1130-10-55) Improving Sensitivity and Accuracy in Nitrite Measurement in Hydroxypropyl Methylcellulose

Purpose: Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low acceptable daily intake limits. One route of N-Nitrosamine formation is from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or the manufacturing of the drug product. One tool available within a wholistic nitrosamine mitigation strategy is to select low-nitrite containing pharma excipients. It is necessary to develop an accurate method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most common pharmaceutical excipients used in drug formulations. The 2D-IC/MS system was selected to overcome two major challenges associated with measuring nitrites in HPMC: sensitivity due to high dilution and coelution (figure 1).

Methods: Nitrite (1000 µg/mL) analytical standard (Inorganic Ventures). ¹⁵N sodium nitrite and sodium hydroxide (95% and 97% respectively, Sigma-Aldrich). Milli-Q high purity water ( >18 MΩ·cm at 25 °C). Sodium sulfate (99%, Fisher Scientific). 100-mL Polypropylene (PP) bottles and syringes/PTFE filters were cleaned with 80 °C sulfate solution and 80 °C water. 1 g of HPMC sample was extracted with 0.1 mmol NaOH at 80 °C for 15 min in a cleaned PP bottle. The sample solution was centrifuged and filtered through cleaned syringes and filters into IC vials for nitrite analysis. A Dionex ICS-6000 dual channel IC system, consisting of AS-AP autosampler with 1000-µL syringe and 8400 µL buffer loop, dual pumps, dual eluent generators, dual ADRS 600 anion dynamically regenerated suppressors and dual conductivity detectors, Dionex auxiliary pump, ISQ EC single quadrupole mass spectrometry and Chromeleon data acquisition/analysis software were used for trace nitrite determination (Thermo Fisher Scientific). The analytical column for the first dimension IC was Thermo Fisher Dionex IonPac AS-24A, 4 X 250 mm and the guard column was a IonPac AG-24A 4 X 50 mm. The analytical column for the second dimension IC was IonPac AS-19, 2 X 250 mm and the guard column was a IonPac AG-19 2 X 50 mm. The 2D-IC system was operated in heart-cutting mode with a concentrator column (UTAC-ULP2 5 X 23 mm) coupling the two dimensions.  Nitrite and interfering peaks from the first dimension were concentrated on the concentrator column and then backflushed onto the second-dimension column for further separation. Nitrite and ¹⁵NO₂^- were monitored at 46 and 47 m/z from 26.5 to 32 min respectively.

Results: The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation (figure 2 and figure 3). MS detection with selected ion monitoring (SIM) and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection (CD). For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. Combination of large injection volume and minimized ion suppression enabled the high sensitivity of the method, which is capable of detect and quantitate nitrite at parts per trillion in sample solution. The optimized method was validated with linearity of nitrite in the concentration range of 18.5 to 5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1% and recovery of 90.5-102.4%. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.

Conclusion: A two-dimensional ion chromatography mass spectrometry method was developed and successfully validated for trace nitrite quantitation in hydroxypropyl methylcellulose (HPMC). Super separation power with 2D-IC separated nitrite away from co-elution peaks and minimized ion suppression in MS. MS detector operated under selected ion monitoring mode solved the commonly encountered interference issue with conductivity detection. The isotope labeled nitrite was used as internal standard to adjust variations in sample preparation and IC/MS system performance to provide rugged performance of the method. This method has the potential to become a universal method for nitrite measurement in all excipients.

Figure 1. Chromatograph showing the coelution issues associated with determining nitrite content using ion chromatography coupled with conductivity detection.

Figure 2. Comparison of nitrite elution profiles of the same concentration internal standard in neat solvent (blue trace) or in HPMC sample solution (orange trace) with (left) CD detection and (right) MS SIM detection. Severe ion suppression with MS detection is observed in HPMC.

Figure 3. Comparison of nitrite elution profiles of the same concentration internal standard in neat solvent (blue trace) or in HPMC sample solution (orange trace) with (left) CD detection and (right) MS SIM detection in 2D. Ion suppression with MS detection was minimized.