Laboratory

The purpose of liquid chromatography is to separate compounds before they enter the spectrometer to prevent overlap (known as co-elution) and increase sensitivity. Before the ionization step, the samples pass through a reversed phase HPLC analytical column where they are separated based on their specific interactions. The column material is called the stationary phase and the solvents used to push the samples through the system are known as the mobile phase.

Important interactions and physio-chemical properties occur between the mobile phase and the stationary phase which causes the analytes to separate and elute at different times. In reverse phase chromatography, compounds that are less water soluble prefer the stationary phase, resulting in a longer retention, while more water-soluble compounds prefer the aqueous mobile phase, resulting in a shorter retention. Before the mobile phase reaches the column, there is also a degassing step that removes unwanted gases (such as air) from the solvents. The steps included in the HPLC process from injection of the sample to ionization are pictured as the first three steps in Figure 2.

The interface between liquid chromatography and mass spectrometry detection is known as ionization. The function of this step involves removing liquid so that molecules of interest are in the gas phase, and ionizing molecules of interest so they may enter the magnetic system of the mass spectrometer. After passing through the column, the analyte ions evaporate from a charged droplet of liquid produced by nitrogen gas and a voltage potential, separating the solvent from the analyte.⁸ The ionized compounds produced from the ion source then passes through the mass spectrometer and detector.

The development of triple quadrupole mass spectrometers allowed the benefits of using multiple mass analyzers to be combined into one, hybrid device, eliminating the need to use more than one instrument for the analysis of one sample.8 Detection using a quadrupole mass analyzer involves various voltages applied to four cylindrical rods that ions pass through for detection. The mass to charge ratio of analytes in the electric field created by the quadrupole will result in the specific detection of analytes of interest only. Three quadrupoles are used in triple quadrupole instruments, each with a different purpose (Figure 2).

The first and third quadrupoles are used as mass filters while the second is a collision region where precursor ions collide with “collision gas”, converting kinetic energy to internal energy and causing the molecules selected in the first quadrupole to break apart or fragment.9 This dissociation allows precursor ions to become product ions before the final filtration step. In quadrupole 1, the precursor ions are filtered through before fragmentation in the collision region, and in quadrupole 3, the specific product ions are resolved based on their mass to charge ratio for final detection. This is visualized in the second half of the steps included in Figure 2.

The following parameters are used for the LC-MS/MS clinical toxicology assay.

• First needle wash: 80% HPLC grade water and 20% isopropanol
• Second needle wash: 60% isopropanol, 20% methanol, and 20% acetonitrile
• Mobile phase A and B: 0.2% formic acid in HPLC grade water and pure acetonitrile
• Methanol blanks between each sample containing 90% HPLC grade water and 10% methanol
• Autosampler temperature: 4°C
• Injection volume: 8 µL
• Analytical column dimensions and temperature of oven: 100 x 3 mm, 50°C
• Elution time for each sample from injection: 10 minutes

Each analyte detected has a corresponding retention time at which it elutes from the analytical column during the assay run time of 10 minutes. The assay used for clinical toxicology testing utilizes a scheduled multiple reaction monitoring mode (sMRM) that will detect analyte peaks only within the window of time where it is expected. This prevents the overlap of peaks and increases the amount of data points that are taken for each peak.

Retention times under the same chromatographic conditions are characteristic for each compound and are used along with the aforementioned ion transition ratios as criteria for structural confirmation. Thus, it is critically important to ensure the HPLC retention times remain constant and as reproducible as possible. As a quality control measure, retention times are tracked daily to ensure the correct window of time is being used for each analyte, and to monitor the shifting of peaks due to issues with one of the instrument’s components.

The most common problems with LC-MS/MS analysis include a loss of sensitivity, decreased peak separation, increased peak splitting, or leaking or clogging of tubing. Another issue faced is the carry-over of analytes from one sample to the next. The maintenance of these highly sensitive instruments is critically important for acceptable sensitivity and accurate results. More detailed quality assurance and quality control guidelines will be outlined further on; here, the upkeep of the instruments alone is discussed. 

Preventative maintenance Many steps are taken daily to maintain the instruments and prevent degradation of the assay or damage to the physical instrumentation and ensure maximum performance. Changing mobile phase solvents regularly and “purging” the instruments so there is no air in the lines eliminates shifting of analyte peaks and maintains accuracy. Monitoring retention times and peak areas can indicate when steps should be taken to improve the quality of the results, such as the following.

Changing the column The analytical column used for the initial separation of samples before entering the mass spectrometer will erode over time and lose its ability to separate analytes successfully. Simply changing the column after every 1000 injections improves separation and sensitivity.

Cleaning the QJet The QJet is responsible for guiding ions in a thin stream from the ionization source to the mass spectrometer in an efficient way. Over time the QJet requires cleaning to ensure ions are able to pass through without issues. One piece in front of the QJet called the curtain plate is cleaned regularly as a preventative measure. Cleaning the QJet and curtain plate improves the sensitivity of the mass spectrometry detector.

Replacement of parts By simply replacing pieces of the LC-MS/MS system, issues such as loss of sensitivity or increased carry-over could be resolved. The injection needle and needle seat, tubing, valves, and more should be replaced before issues arise. Tubing and valves that liquid samples pass through will become clogged or erode over time and are replaced on a regular basis to avoid any problems.

Expired analytes Ensuring the quality of analytes used as reference materials and in the internal standards, including that they are used before their expiration dates is critical for calibrators, quality control samples, and for the accurate quantification of analytes in patient samples. Internal standards are important for quantitative data analysis to correct variations and ensure repeatability between samples.⁸ In order to determine the unknown concentration of an analyte in a sample, a known concentration in the internal standard is required. As analytes expire, the peaks detected will lose intensity and split, causing questionable accuracy of patient sample results, deeming them unacceptable.