Development and Validation of a Sensitive LC-MS/MS Method for Simultaneous Determination of Several Nitrosamines in Large Volume Parenteral

Abstract:

Background

Nitrosamines have gained significant attention in pharmaceutical industry. However, due to the significant daily dosage of large volume parenteral (LVP), the detection limit for these products should be in the ng per liter range, which many published methods cannot achieve.

Objective

This study aimed to develop and validate a sensitive LC-MS/MS method for the simultaneous determination of several nitrosamines in LVP drug products.

Method

Nitrosamines and related internal standards were separated on a Waters ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 µm) column on a LC-MS/MS system with gradient elution. Prior to the LC injection, a Carbon A solid phase extraction (SPE) column was utilized to pretreat the test solutions. The mobile phase was composed of 0.1% formic acid in water as mobile phase A, and neat methanol as mobile phase B. Analytes were detected via multiple reaction monitoring (MRM) mode and quantitated against internal reference standards with a quantitation limit of 2.5 ng/L for N-nitrosodimethylamine (NDMA), 0.75 ng/L for other Nitrosamine analytes.

Results

The LC-MS/MS method was able to separate all analytes of interest by gradient elution within 15 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline ICH Q2(R2). The LOQ for NMDA is 2.5 ng/L, while for other nitrosamines, it is 0.75 ng/L in peritoneal dialysis and saline matrices. For haemofiltration solution, the LOQ is 1.0 ng/L for NDMA and 0.3 ng/L for other nitrosamines. The RSD% (n = 9) of the recovery did not exceed 25% in the method accuracy evaluation. A comparative testing in three labs revealed that all three laboratories are capable of accurately measuring nitrosamine levels at 10 ng/L within the intricate LVP matrix.

Conclusions

The LC-MS/MS method for several nitrosamines was successfully developed, validated, and demonstrated to be accurate, robust, and specific.

Highlights

The performance of this method can reach single digit ng/L level in LVP matrices. Comparative testing was conducted in three laboratories located in China, Germany, and the United States to ensure reproducibility of the method.

Nitrosamine impurities, trace level analysis, full method validation, LC-MS/MS, large volume parenteral (LVP)

Accepted manuscripts

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