We have proceeded in a complete acid degradation process of lercanidipine drug and we separated the degradation products by HPLC to obtain 5 degradation peaks, then for further identification and estimation of these 5 degradation products, we used UPLC/MS which gave only 3 peaks. We couldn't find the missed two peaks of the two remaining degradation products.
Hi Shereen,
Thanks for the question. I think a bit more information is needed for a full answer and will set it out below to try and help.
You degraded a drug and got 5 separate peaks, I take it this was using HPLC-FLR/UV detection? I take it you had good separation and resolution of these peaks?
You did the same assay by LC-MS but could only see 3 peaks? Could your products have degraded before your LC-MS analysis? A lot of degradation products can have short stability.
If you are sure that they haven't degraded then I would suggest swapping out your current LC unit connected to your MS if it doesn't have an FLR/UV detector. This way you can have a confirmatory chromatogram using UV before going into the MS. If you still see the peaks in the FLR/UV detector then your issue is in the MS.
On the MS side of things it is a bit harder to troubleshoot with the information given. By the sound of it you will be running a full scan in Q1 at the respective retention times for the masses of the products.
Are you using electrospray ionisation (ESI)? APCI? Or another ionisation technique? ESI is generally used as a first approach. The problem being is that this can be quite harsh on compounds being ionised, especially if they are low mass (such as some of your degradation products). I would possibly try a softer ionisation technique to see if this improves.
Another problem might be that the products you can't see may be only visible in the opposite polarity. An easy test for this is to run your sample in positive mode and again in negative mode. If you see the other 2 products then you can make a single method with a positive/negative switch for these peaks.
I hope this helps to troubleshoot your issues and gets you on the right path.
You asked if the number of peaks seen in a MS plot might differ from those seen in some type of other chromatogram (detector not specified)? Quick answer, YES.
First, terminology... HPLC is the technique and "UPLC" is a trade-name used by one company for their HPLC. We prefer to use the correct scientific names, not trade-names to avoid confusion (Always referred to as "HPLC"; https://hplctips.blogspot.com/2015/08/terminiology-which-is-it-uplc-uhplc-or.html).
Next, It sounds like two different HPLC systems with different detectors were used for the analysis (#1, HPLC and one detector (?), then #2, HPLC with a MS detector) . Again, "HPLC" is a name for an analytical technique alone, not a method. HPLC must be used with a detailed method, to create an "analysis method" (this is what must be shared when discussing questions). HPLC by itself is just a tool (it does not do anything unless it is combined with a complete method and their are millions of possible methods). All HPLC systems also need a detector to provide data output. There are many types of HPLC detectors available for this purpose. UV/VIS is the most popular, but RID, FLD, ELSD, CAD, ECD and various types of mass spectrometers (MS) are also available. Depending on the method used and sample's physical and chemical properties, some detector types may or may not be appropriate. We do not know what type of method or detector were used to develop the first method used on your samples (probably a UV/VIS ?).
- UV/VIS detectors do not "see" everything as this depends on how well the sample absorbs light. Some do not absorb at all. So with UV/VIS you may detect items which in fact are extremely low in actual concentration, but appear HUGE in the chromatogram. Their response is different for different compounds.
- The same problems exist with all types of MS detectors too. MS detectors Do Not Detect everything. They only detect compounds that ionize well under the method and instrument conditions used. So a MS detector will not "see" many types of compounds. Their response is different for different compounds.
We often setup and run an HPLC system with both a scanning UV/VIS (DAD) detector inline with an appropriate type of MS detector. This allows us to collect orthogonal data at the same time on all peaks detected. The two types of data are complementary and when review by an experienced operator, can help us better understand what we are looking at.
The result is that the same sample analyzed on two different detectors (using the same exact method on the same exact HPLC system) will often show UV/VIS peaks that have no MS data and MS peaks with no UV/VIS data (normal).
Two possibilities, peaks may get merged and co-eluted, or they may be further degraded.
Its very Rare. Two very closes eluting may get merge or Seperate. Its becuase of mixing in pump occurs. But Changing in Gradient composition same Pattern can be achieve.
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