Liquid chromatography-mass spectrometry or LC/MS is a technique to analyze different kinds of chemicals. It is a combination of HPLC (high pressure liquid chromatography) with a UV detector and mass spectrometer. At Emeryville Pharmaceutical Services (EPS), the LC/MS has an additional detector, an Evaporative Light Scattering Detector (ELSD) connected.
Liquid chromatography is a primary technique to separate different compounds in a solution based on their chemical properties such as their hydrophobic characteristic (reverse-phase chromatography), their sizes (size exclusion chromatography), or their charges (ion exchange chromatography). This is done with the help of different columns and solvents.
After separation, the compound is analyzed by going through the detector system. First, the eluant will go through the UV detector where compounds which absorb light will be detected. Each molecule can absorb light at a certain range of wavelength. We can optimize the wavelength to get better resolution.
After going through the UV detector, a portion of the eluant will go to ELSD and other portion will be directed to MS detector. Mass spectrometry was established based on the ability of molecule to be ionized by a dual ESI/APCI ionization. ESI is an electron spray ionization method where the small droplets of the analyte will be ionized. APCI or atmospheric-pressure chemical ionization can ionize the compound in gas phase. Both ionization sources run simultaneously on our system, maximizing chance of detection by MS.
ELSD detects non-volatile compounds. The technique was developed based on the ability of particles to scatter light. After the flow of solvent and the target molecule is nebulized, only the solvent will be evaporated, so all the target compounds will remain in the system and be dried to become small particles. Finally, a source of light is shined through these particles and a photomultiplier detects all the light scattered by the particles.
Having ELS detector makes our LC/MS more powerful because it can detect a wider range of compounds. With the compound that has low light absorbance, it is difficult to detect the compound with PDA detector because of the low peak intensity. In that case, ELSD will be useful to detect it. Moreover, the result does not depend on the solvent since all the solvent is evaporated. Thus, it does not scatter light which means there is no solvent peak appeared at the beginning of the run as it does in PDA chromatogram. In addition, the ratio of signal to noise is minimized especially with a gradient elution because the baseline will not drift.
As EPS has been offering LC/MS services, we always want to improve our technique to better assist the scientists. This project aims to compare methods of determining unknown concentrations using ELSD, UV, and MS detectors.
Elaboration of ELSD method to determine unknown concentration:
Fmoc-Asp-OH (MW 355.35 g/mol) has been used as a standard for LC/MS because it absorbs UV, gives a robust ELSD signal, and ionizes in both positive and negative mode.
A dilution series starting from 10 mg/mL of this standard was prepared and run under our standard LC/MS method. The experiment was triplicate to minimize the error during sample diluting.
The area peaks of UV, ELSD and MS detector were recorded. The data generated was used to compare the accuracy of ELSD method to those of UV and MS.
MS signals were registered under both positive and negative modes which gave 2 base peaks of respectively m/z 354 (neg, [M-H]–) and m/z 378 (pos, [M+Na]+). The intensity of the base peak was read by both TIC (total ion count chromatogram) and XIC (extracted ion chromatogram) modes. TIC mode counts all the ions in the certain range of masses. In contrast, XIC mode allows measuring the intensity only of the molecule of interest.
The ELSD signal recorded from LC/MS shown that the ELSD works best with a concentration in the range of approximately 0.025 mg/mL to 2.5 mg/mL. Indeed, the ELSD chromatogram of the concentration 0.01 mg/mL (less than 0.025 mg/mL) resulted in a small peak which is indistinctive from the background. Also, the ELSD is saturated at concentration of 5.0 mg/mL.
A log (ELSD integral) vs log (concentration) was plotted (fig 1) which generated a linear trendline with a R2= 0.99. The error bar is likely consistent at every point which is good for the result liability.
Next, UV signal, MS with ESI (+) and ESI (-) signal using TIC and XIC methods of each concentration were also plotted ( fig. 2, 3, 4 respectively) to compare with the ELSD result.
The UV signal increases proportionally with the increase of the concentration of the sample. In fact, the trend line has a high R2 value around 0.99 and the error bars were relatively smaller than those of ELSD signal. The fact that this line goes through the origin makes the experiment more valuable. Nevertheless, the concentration that UV can detect was 0.05 mg/mL to 2.0 mg/mL, meaning this method has a higher lower limit of detection than ELSD.
Both ESI (+) and (-) signal gave 2 trend lines that did not go through the origin. Moreover, the error bars are not consistent. The range of the concentration that MS can detect started at 0.2 mg/mL which is less sensitive than ELSD. (Fig 3, 4)
The XIC mode gave a better signal than TIC mode to quantify a compound. From the figures below, the XIC mode gave a linear curve with R2 about 0.99 whereas the linear curve was far off in case of using TIC mode. The XIC mode is more effective because we can integrate the peak area of only the m/z of interest.
Relationship between the molecular weight and the ELSD signal:
It has been known that the molecular weight of a particle can affect the ability to scatter light. Clarithromycin and N-acetyl-DL-methionine were intentionally chosen to examine the behavior of ELSD signal with response to the changes in molecular weight. Clearly, the clarithromycin has a molecular weight of 747.95 g/mol, which is twice higher than that of Fmoc-Asp-OH. Meanwhile, N-acetyl-DL-methionine has a relatively low molecular weight of 191.25 g/mol. These compounds were prepared with a similar dilution series to the one of Fmoc-Asp-OH.
The trendline of the Clarithromycin was likely superimposed to the one of Fmoc-Asp-OH while the trendline of N-acetyl DL methionine is slightly slow on the logarithmic scale. The fact that they are not entirely overlapped confirms that the photon scattering depends on the molecular weight. Nevertheless, they still generate linear curves with a high R2 value which is very promising for ELSD signal method.
|Concentration range (mg/mL)||0.05-2.0||0.025-2.5||0.2-2.0|
|Accuracy||Most accurate||Accurate||XIC mode accurate TIC mode accurate|
Based on these data, we now confidently offer the ELSD method to quantify an unknown compound given a standard. By plotting a standard curve of the compound of interest, we can quantify its concentration. The UV signal is the most linear and most reliable for quantitation. However, the range of concentrations which can be detected is better for ELSD than MS and UV. In the case where a compound submitted is in small amount, ELSD would be a better choice.