In detail
How it works
In the liquid chromatograph, the components of a sample are separated in time according to their interaction with the column. The separated substances are then ionised and enter the mass spectrometer. In a triple quadrupole instrument, the first mass filter selects the ion of the target substance, a collision cell fragments it, and the second mass filter only lets a specific fragment pass. This technique is called multiple reaction monitoring (MRM). The combination of retention time and mass transition makes the measurement highly selective.
Use in pharmaceutical testing
Trace impurities that UV detection cannot capture with sufficient sensitivity or selectivity.
Nitrosamines: EMA requires mass-selective techniques such as MS/MS or high-resolution mass spectrometry for their determination, because co-eluting substances can otherwise cause false results. It cites DMF, which can co-elute with NDMA, as an example.
Quantitative determinations in complex matrices where other techniques reach their limits.
Validation under ICH Q2(R2)
ICH Q2(R2) includes quantitative LC/MS analysis of trace impurities as a dedicated example. Specificity can be justified on technological grounds, for instance by the MRM transition with a specified quantifier to qualifier ion ratio in combination with retention time. For precision, the guideline gives at least three replicates at each of at least three spiking levels or six replicates at 100 per cent, and for linearity at least five concentration levels. Intermediate precision is tested in the same laboratory under varying conditions, for example with different LC/MS systems, different analysts or on different days. The quantitation limit is the lowest spiking level with acceptable accuracy and precision.
Common pitfalls
In trace analysis, contamination from water, laboratory air, gloves, membranes or solvents can cause false positives, and nitrosamines may even form during the analysis itself. EMA therefore recommends control experiments, for example using an orthogonal method.
In practice at A&O Pharma
In our GMP-certified laboratory in Itzehoe, we use triple quadrupole LC-MS/MS for trace analysis. Analytical method validation is part of our GxP consulting, so testing and validation can come from a single provider.
Analytical ServicesFrequently asked questions
What is the difference between LC-MS and LC-MS/MS?
In LC-MS, the ion of the substance is measured after a single mass stage. In LC-MS/MS, this is followed by fragmentation and a second mass stage, which markedly increases selectivity and reduces interference from other components.
Why is LC-MS/MS used for nitrosamines?
Nitrosamines have to be determined at very low concentrations. EMA requires mass-selective techniques such as MS/MS or high-resolution mass spectrometry to avoid false results from co-eluting substances.
How is an LC-MS/MS method validated?
In line with ICH Q2(R2), which includes quantitative LC/MS analysis of trace impurities as a dedicated example. Specificity, precision, accuracy, linearity and the quantitation limit are among the characteristics assessed.
