Guide to Troubleshoot Your LC-MS Assay Method

Liquid Chromatography-Mass Spectrometry or LCMS is a combination of two different selective techniques. The technique allows simultaneous isolation and measurement of the analyte of interest for the mixture of the complex compounds. The LC part of this technique helps in differentiating compounds based on their physicochemical parameters. The MS part separates them according to their mass to charge ratio. The dual selectivity of the LC-MS technique helps in making it a powerful analytical tool. 

Troubleshooting Tips for Typical Failure Causes – 

The prime objective for troubleshooting of the LCMS unit is providing the readers with the required optimization information for overcoming the issues related to disrupted quantification. Here are specific troubleshooting parameters – 

Retention Capacity – 

Start by reducing the carbon load of your LC MS analysis column; e.g., if you are using a C18 column, start using C8. Prefer using a stationary phase that is bonded to the same silica type, which helps ensure consistent secondary interactions. 

Lack of resolution caused by an insufficient selectivity – 

When the optimization of the C18 or C8 column is not possible, you can try columns supported with a higher polar stationary phase. 

Carryover and Contamination – 

You can work out the carryover contaminant estimation by obtaining the response from the blank injection after analysis of the desired standard solution. 

Preparation and Storage of the Calibration Standards – 

The solvent to be used for calibration standards should be tested for their solubility, stability, and volatility. 

LC-MS calibration – 

Prepare the required calibration solutions by using a matched matrix or pure solvents. This prepared calibration solution can be used for LCMS’ external calibration. 

Other aspects of LCMS calibration – 

  • The essential requirement for the number of calibrated levels designed for method validation and batch analysis
  • Required concentration levels for the calibrants taken into consideration by bearing the sample concentration levels in mind
  • The counts of the replicate measures required both for calibrants and samples. 

Method Validation – 

The following points are crucial in designing your LCMS method validation protocol – 

  • Method validation should support the assessment of the performance of the LCMS technique, including everything from measurement procedure to sample preparation to the end measurement of the LCMS. 
  • The primary aim of evaluating the performance of the developed LCMS method should be inclusive of the material analysis, which represents test samples and materials possessing a known value. 
  • The LCMS method validation should always be inclusive of evaluation of the uncertainty in measurement values. It should be optimized for its intended purpose. 
  • The most common performance parameters evaluated for the LCMS method validation are selectivity, analysis of the replicate precision, biases, limit of detection, the limit of quantitation, linearity and working range, ruggedness, and robustness. 

Measuring uncertainty – 

The uncertainty in the measurements of LCMS analysis is usually calculated for determining the individual test results. Instead, the degree of uncertainty for the conventional test materials is evaluated, which is later applied to all remaining results, which is obtained when the method is accomplished correctly within its specified scope. 

These are the basic troubleshooting parameters for your LCMS units. 

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