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The Failure of LC-MS Bioanalytical Research Method

2021-11-25
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A successful LC-MS bioanalysis method transfer can increase the confidence of the sender and recipient laboratories. However, because the transfer of LC-MS bioanalysis methods involves preliminary preparation, communication, and acceptance, and the content of each process is numerous, the negligence of individual details during the method transfer may cause the transfer to fail, and the reasons after the failure Analysis has become a top priority. This issue summarizes the reasons for the failure to transfer the LC-MS bioanalytical research.

Reason 1: The laboratory settings of the sender and the receiver are different

A successful method transfer depends not only on the experience and knowledge of the sending and receiving laboratories in regulating biological analysis, but also on the understanding and communication of the characteristics of the transferred method. The two laboratories are engaged in HPLC systems, mass spectrometry, pipetting methods, automatic pipetting systems, reagents, reagent storage, plate washers, glass tube siliconization methods, the use of special equipment, vortex mixers, and even air flow in fume hoods, etc. The slight difference in the method may lead to the failure of the method transfer verification. Therefore, it is very important for the sending and receiving laboratories to clearly communicate the details of the method before the method is transferred.

Reason 2: The stability of the substance

The stability of the analyte or its metabolites can also affect the repeatability and accuracy of sample analysis. There are many factors that cause the instability of the test compound or its metabolites, including enzyme-catalyzed degradation (such as ester-containing drugs or prodrugs), chemical reactions (such as thiol compounds), auto-oxidation of catechols, lactones, and Interconversion of hydroxy carboxylic acids (such as statins), degradation of nitrogen oxides and degradation of phase II metabolites, chiral/epimerism/tautomerism/isomerism interconversion, etc. Among the above reasons, the metabolites of phase II conjugates (such as acyl glucuronic acid) degrade into the parent analyte during sample storage, processing, or chromatographic analysis. Degradation of the unstable phase II conjugate will undoubtedly lead to an overestimation of the concentration of the parent compound and an obvious positive deviation when the analyzed sample is reanalyzed in the receiving laboratory. It is important to review the stability of the analyte and metabolites and test the relative stability. If the analyte does contain unstable metabolites, additional stabilization measures should be taken for sample collection and processing before liquid chromatography-mass spectrometry (LC-MS) analysis. Any findings should be clearly explained to the receiving laboratory.

Reason 3: inappropriate method selection

The evaluation of analytical method selectivity should use 6 sets of LLOQ concentration samples prepared with different matrices. Among the 6 LLOQ concentration samples, the deviation value of the 5 analysis results should be within ±20% of the labeled concentration. For some difficult-to-obtain matrices, less than 6 sets of matrices can be used for selective testing; before transfer, even a set of combined matrices can be used for testing. Testing a single set of matrices can mask the differences between different sets of matrices, which can lead to failure of method transfer in the case of poor sample analysis selectivity. In addition to some common endogenous components (such as phospholipids), known or unknown metabolites, dosing vehicles, and drugs taken at the same time, will lead to insufficient analysis selectivity.

Bioanalytical Lab

The main basis for chromatographic separation of analytes is the difference in physical and chemical properties of the analyte and matrix components relative to the mobile phase and the stationary phase on the HPLC column. When the mass spectrometer is operated in single-reaction monitoring or multiple-reaction monitoring (MRM) mode, there will be less interference. Atmospheric pressure ionization techniques [such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)] are generally regarded as “soft” analytical techniques. However, any molecule with weak bonds can be cleaved during the ionization process before entering the Q1 chamber of the tandem mass spectrometer (in-source cleavage). This “in-source lysis” is especially obvious if there are metabolites such as nitrogen oxidation, sulfur oxidation, glucuronic acid or sulfuric acid conjugates of the analyte in the biological sample extract. In-source cleavage of these metabolites can produce the same ions as the precursor ions of the parent compound. In high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the chromatographic separation effect is often affected by the greatly shortened liquid chromatographic separation time. If the chromatographic conditions of the sender’s and recipient’s laboratories are different, in-source lysis of metabolites may cause significant differences in the concentration of the tested sample measured by the sender’s and recipient’s laboratories. In this case, it is often necessary to modify the chromatographic method in one or both laboratories in order to re-validate the method.
Reason four: common experimental operation errors

The failure of the reanalysis of the tested sample (IS R) may not be caused by the problem of the analysis method itself (selectivity or stability problem), but by the error of the experimental operation. These errors include but are not limited to: ①The sample is not sufficiently melted, ②The sample is not uniform before sampling, ③The sample is improperly diluted, ④The sample identification is unclear. In view of this, the correct training of laboratory personnel should be ensured.

Investigation of method transfer failure


The failure of any method transfer should be investigated as necessary. In this regard, the sender and recipient laboratories must have a written procedure approved by both parties (such as a method transfer agreement or an agreement amendment).

Generally speaking, the investigation will consist of two stages: stage I, reviewing existing data and documents; stage II, laboratory investigation, both sender and receiver laboratories may need to be conducted.


In the Phase I investigation, document review should focus on sample identification, preparation, data processing, errors in the calculation process, or possible incorrect settings in the LC-MS system. At the end of Phase I investigation, there should be the following 3 possible results:

① A clear cause is found, that is, the root cause of the method transfer failure is confirmed;
②The possible reason is found, that is, the reason for the method transfer failure may be confirmed, or be narrowed down to several possible reasons;
③ No clear cause is found, that is, the direct cause cannot be confirmed.

If an attributable cause is found, it should be corrected immediately. Sometimes a simple correction in data processing and calculations may end the investigation. Of course, necessary relevant training should be carried out to prevent similar situations from happening again.


In the event that one or more possible laboratory operations or instrument errors are found, a Phase II laboratory investigation should be carried out to confirm the root cause. Based on the results of the test, the following judgments can be made:

① The attributable cause is confirmed;
②No cause to blame has been found.

Regarding the previous point, various work should be started immediately, including but not limited to re-analysis of samples. For the latter, a more in-depth investigation should be carried out, and if possible, the sending laboratory should also carry out a corresponding investigation. When the method is carefully reviewed, the possible effects of the following factors should be evaluated: inappropriate sample preparation, unknown unstable metabolites, unsatisfactory chromatographic separation of analytes and endogenous compounds, unsatisfactory method selectivity, etc. One or more experiments should be performed to confirm the possible root cause. This investigation may lead to a simple step correction and sample re-analysis, or it may lead to the redevelopment and re-validation of bioanalytical methods.

Depending on the purpose, the transfer of a bioanalytical method may be as simple as a single batch cross-validation, or it may be a comprehensive validation. With the current trend that the pharmaceutical industry will increasingly outsource post-drug R&D work to CROs (especially CROs in emerging markets), comprehensive method validation may become necessary. In any case, the results of the method transfer should meet the requirements of the drug regulatory authority. Therefore, both the sender and recipient laboratories should work together. All technical challenges should be discussed through appropriate channels, including field visits or training. The recipient laboratory should not be afraid to question the transferability of the method.

Finally, a successful method transfer can not only reduce the cost of drug development, but also help the two laboratories to establish a long-term cooperative relationship and achieve a win-win situation.

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