A technology frequently used for analytical detection is chromatography with mass spectrometry. Liquid chromatography (LC) and mass spectrometry (MS/MS) components make up LC-MS/MS. The targeted compound is extracted from the sample by the LC and then split into smaller pieces for identification and quantification by the MS.
Though Chemists may be routinely familiar with the LC-MS analysis, the concerns and operations involved in drug development methods for compound optimization are different. This article offers points to be kept in mind for compound optimization for successful LC-MS method validation.
Dilution of chemical standard A chemical standard devoid of other compounds is required if we wish to teach the instrument of an unidentified substance. The bar is diluted to a reasonable concentration (50 ppb-2 ppm) with a suitable solvent depending on the instrument sensitivity.
Early drug development: After deciding on a target, the pharmaceutical industry and, more recently, some academic institutions have streamlined several preliminary procedures to find compounds with the necessary properties to produce acceptable medications.
- MS/MS Optimization
The MS/MS process primarily contributes to the signal deduced from the spectrum. We must first collect the MS/MS characteristics of the target compound to confirm that the signal we eventually obtain is coming from our target compound and not something else running out of the LC.
3. Optimization of the ionization energy of the parent ion
To optimize the parameters for the parent ion, we need to know its mass. As a rule of thumb, mass is usually its molecular weight plus or minus a proton from the original molecule ([M+H]+ or [M–H]– ).
4. Optimization of collision energy of the fragmented ion
Different spectra are created upon forming other fragments under differing collision energies. The most prevalent pieces can be found by scanning a variety of collision energies and overlaying the spectra that result or “daughter ions.”
5. During biotech drug development, a compound is said to be found if
a. It contains both MRM pairs as with the standard
b. The MRM pairs have the same ratio as the standard
Therefore, it is advised that at least two MRM pairs be optimized for each compound. The mass of the daughter ion might occasionally match that of a commonly used solvent or be sensitive to interference during sample preparation.
- Chromatography optimization
The next stage is to optimize the LC conditions after the MS/MS compartment is optimized. Due to competition between the stationary and mobile phases, LC separation depends on the compound’s retention. The physical and chemical characteristics of the target substance should be considered while choosing a column.
- Verification with a calibration curve
LC MS verification: Verify whether the optimized conditions are valid for the targeted compound before beginning sample preparation. Using solutions with different compositions or conducting a test using a calibration curve are two methods for verification.
8. Amount of sample required
A small quantity of the compound is necessary for mass spectrometry. If the instrument is susceptible, you can use concentrations lower than 1 mg/ml.
9. Storage of stock solutions
In a refrigerator set to 2°–4°C, a stock solution of macronutrients is stored for several weeks without risk. Micronutrient stores should be kept in a freezer or refrigerator until used.
“Carryover” refers to the phenomenon in which analytes from one run of the LC-MS system continue to exist and get detected in the subsequent test. After sample analysis, the carryover is verified by measuring a blank solution, such as water.