Challenges and winning strategies to develop an analytical method for Nitrosamines

Problem source

Challenges

Way forward

Peak shape and separation

Sample matrix, high reactivity components and low response of analytes

1) Different Sample preparation strategy extraction and filtration.

2) Diluent compatibility, pH of Eluent and diluent. 
Separation of closely eluting components by selection of stationary phase.

The chemical diversity

Highly polar impurities result in very low retention & poor reproducibility.

1) Selection of stationary phase and addition of an ion-pairing agent to the mobile phase.

2) Selective Hydrophilic interaction liquid chromatography mode.

High eluent pH

High eluent pH (> 10) needed for a photochemical reaction which limits the sensitivity and complicates the analysis procedure.

1) Addition of an anion exchange module to the HPLC.

Sensitivity

Adequate sensitivity

1) Required Sensitivity can be achieved by cleaning the sample matrix using sample treatments procedure.

2) Suppressing unwanted matrix by applying compatible sensitive detection techniques: UV detection, evaporative light-scattering, refractive index, fluorescence detector, (LC with mass spectrometer) MS-MS, HRMS & MALDI imaging mass spectrometry.

Solubility and matrix

Solubility of target analyte and unwanted complex matrix components.  

1) Selection of diluent for target analyte.

2) Efficient extraction procedures followed by matrix spiking recovery study.

Structurally similar impurities.

Coelution of impurities with target analyte and poor resolution between closely eluting impurities.  

1) The Approaches and Considerations for choice of stationary phase, pH modifier, column temperature and organic solvents are important to prevent coelution of peaks and enhanced the resolution.

2)& Specifically separation of Target analyte and impurities NDSRI

Cis and Trans Nitrosamine analysis, interpretation & reporting  

The secondary amine protonated, and it will be tautomerized to

N = N leads to form cis-trans isomers. Isomer’s abundance based on intramolecular hydrogen bonding capability and the hindrance level of each isomer to create a more thermodynamically stable form.

Report as a sum of cis and trans

 Detection limit & Simultaneous Quantification.

Challenge due to the problems of high detection limits and difficulty in simultaneous N-nitrosamine compounds detection.

1) Efficient n-hexane pretreatment to remove non-polar impurities before the conventional process of column extraction.

2) Efficient and robust sample treatment Ex. Solvent extraction will enhance the reproducibility and recovery parameter of the method.

Selective Quantification of each nitrosamine.

Sensitivity and selectivity of the method.  

1) ESI (+) mode to get better signal response along with Selection of MRM.

2) Automatic optimization to obtain the parent ion, daughter ion, optimal cone voltage and collision energy of the target compound.

Matrix effect

 Presence of bulkier groups in API & number of related impurities.

1) The compatible sample preparation workflow including spiking, extraction, sonication, centrifugation followed by the filtration of supernatant aliquot by using 0.45 µm PVDF filter.

2) These sample preparation flow will reduce the matrix effect as well spiking will ensure the analyte recovery throughout the sample preparation flow.

Sensitivity, precise, and accuracy.

Poor chromatographic retention, MS ionization, and fragmentation, often limiting sensitivity and selectivity.

1) APCI MS operating in the positive ion mode improves the ionization of target analyte.

2) MRM transitions and optimization work for nitrosamine analysis.

3) An improvement in signal can be achieved by optimizing probe and source temperatures increase the response of nitrosamine impurity.

4) These optimizations and tuning improves the Sensitivity, precise, and accuracy.

Early eluting matrix (salts and excipients)

Close elution of Nitrosamines to the solvent front with the salt.

1) Use the fully end capped, high coverage C18 phase column retain the smaller nitrosamine impurities, the 𝜋-𝜋 interactions of nitroso groups with the stationary phase.

2) The primary and secondary interaction with method parameter optimization improves the retention and resolution.

SIM and SRM

Lowering of sensitivity using SIM

1) Multiple reaction monitoring (MRM, also known as Selective Reaction Monitoring – SRM) is a highly specific and sensitive mass spectrometry technique that can selectively quantify compounds within complex mixtures.

Problem source

Challenges

Way forward

High Injection Volume

To achieve the sensitivity large volume of a test sample is introduced into the GC column and it can get damaged in the long run.

Headspace sampler techniques is preferred along with to avoid the backflush injector temperature to be optimized.  

Inlet temperature /

Thermal Stability

Setting the inlet temperature too high can lead to thermal decomposition of the analytes and matrix, potential thermal degradation of analyte and drug substance.

The LC. UPLC also enables the simultaneous determination of nitrosamines alongside drug product in a single run.

Sensitivity of Detector (FID)

Sensitivity of a flame ionization detector is not sufficient for some compounds.  

The Pre-column derivatization technique is required before carrying out the analysis. Derivatives should be thermally stable.

Diluent Matrix effect.

DMSO and coeluting analytes may increase the SIM signal. DMSO’s boiling temperature is high and like that of the GTIs, triggering the effect of the diluent matrix.

Alternative diluent like polar and mid polar solvents.  

Physicochemical properties:

GTIs are non-volatile, highly reactive, acidic, and non-chromophore and causes a problem for analysis and identification of drugs.

Consider the compatibility of stationary phase, elution, sample preparation and detection strategy.

 Robustness issues

Potential interferences resulting from large quantities of drug substances and their impurities.

Sample preparation strategies to eliminate the matrix effect, enhance the analyte signal and robustness of the method.