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The list of articles:
#2 Two myths about ion HPLC of pharmaceuticals >>
#3 Medical cannabis fever: what an ideal HPLC separation should look like >>
#4 Taming HILIC: two advices to develop a perfect pharmaceutical application >>
#5 Why not ion exclusion: analyzing pharmaceutical combinations >>
#6 Drug screening: group selective HPLC stationary phases are the choice >>
It is a common point that peak tailing in reversed phase HPLC mode is always a problem for basic pharmaceuticals. So many words were told and written on this topic!
However, the solution is quite straightforward. Use moderately acidic mobile phases, use moderately concentrated inorganic buffers for mobile phases, use high-quality HPLC columns - and that's it. I usually use 50mM ammonium dihydrogen phosphate buffer + 0.1% or + 0.5% phosphoric acid (pH of the last one is around 2 which is Ok). You can try something else of this kind.
And if your target compounds are simply amines or beta-hydroxy amines, and you observe excessive peak tailing, this means you've got a bad HPLC column. You should face it. Stop wasting your time, just find an appropriate HPLC column for the given separation.
Please, look at the HPLC separation of different antihypertensive drugs (α-blockers, β-blockers, Ca channel blockers, diuretics) obtained in reversed phase mode on IBSpharm AH-1 HPLC column. All target compounds are amines. Do you see any peak tailing? No. However, it does not mean this column is unique. It simply means that it is good enough for this RP separation.
The overall conclusion is that using only high-quality HPLC columns for HPLC method development does make sense.
#1 Off the agenda: peak tailing for basic pharmaceuticals is not the issue anymore
The first common myth is that it is nearly impossible. You will get miserable plate count, peak symmetry will be awful, HPLC columns will die one at a time every two weeks, and so on and so forth.
But please look at the figure. You see the HPLC separation of different basic bronchodilator, antitussive and antispasmodic drugs in ion HPLC with excellent plate count and symmetry. The column I used for this separation was IBSpharm MA-1 specialty HPLC column, and it was the 3-year old one, and its performance remained the same as for the new column.
So, what's the catch?
For silica-based ion-exchange columns use not less 50% AcN content in a mobile phase, do not use salt gradients and, of course, use only high-quality HPLC columns - that's it.
The second myth is that ion HPLC is not compatible with MS detection because of salt in a mobile phase. So, try C18/WCX or C18/WAX column. Their capacities are so low that it is no problem to obtain ion HPLC separation with no salts in mobile phase at all.
The overall conclusion is that ion HPLC is the most undervalued HPLC technique for the pharmaceutical industry.
#2 Two myths about ion HPLC of pharmaceuticals
#3 Medical cannabis fever: what an ideal HPLC separation should look like
Here we will consider what an ideal HPLC separation should look like through the example of medical cannabis compounds separation in isocratic elution mode.
Please, take a look at the figure. You see the HPLC separation of eight cannabinoids using IBSpharm CN-1 column: two acidic compounds (CBDA, THCA), four monophenols (CBN, CBC, delta9-THC, delta8-THC) and two diphenols (CBD, CBG). Delta9-THC and delta8-THC are isomeric compounds.
There are two critical pairs in this separation: CBD/THC8 and THC9/CBG. They are placed at the end of the chromatogram, which makes their resolution exceptionally robust and unaffected by extra-column voids and other peak broadening factors such as overloading, injection in a ‘bad’ diluent, lost of column plate count during its long-term use, etc.
This separation can be used also for stability indicating methods since it provides good resolution of target compounds from various degradants that might appear in cannabis samples due to their contact with air oxygen. The most of degradants elute either between CBC and CBD or after CBG, which is not a problem because of much space there. But on all types of RP columns (including this one) the one major degradant always appears between delta9-THC and delta8-THC. Therefore, these two isomers should be separated quite well to provide enough space to place a peak or two between them, which the present column does.
Next, a separation should withstand overloading the column with CBDA and THCA, since they are the major compounds of real medical cannabis samples, and also large volume injections in ‘bad’ diluents such as methanol and 2-propanol. This requires:
- isocratic elution, because overloading effects are the most pronounced in gradient elution mode;
- using long columns packed with fully porous particles;
- enough space on the chromatogram around CBDA and THCA peaks.
None of the existing HPLC solutions developed by vendors (such as Agilent, Shimadzu, Restek, Perkin Elmer, Waters, etc.) or by independent researches are suitable for this term. For example, on conventional C18 stationary phases overloading a column by CBDA usually leads to its coelution with CBD and CBG, and thus ruins the separation.
On the contrary, the present column can be easily overloaded with CBDA and THCA without adversely affecting the HPLC separation.
Finally, this HPLC separation is group selective, which means that elution windows for analytes belonging to different chemical classes do not overlap. In the present case, all phenolic acids elute before phenols, which facilitates identification of cannabinoids, and thus makes the separation quite appropriate for researchers.
1. If you see some HPLC separation of standards with formally resolved peaks, it does not mean you will have good chromatography in case of real samples.
2. Vendors not always develop good HPLC applications.
#4 Taming HILIC: two advices to develop a perfect pharmaceutical application
The technique previously known as liquid-liquid HPLC became popular in 80’s under the name ‘HILIC’ as a promising tool for separating and isolating various biopolymers.
It has been flourishing in 2000-2010, but the great interest in this method has suddenly waned in the recent decade by contrast with the significant overall growth of pharma and biotech HPLC applications. So, what happened, and is it possible to reverse this trend?
All HILIC problems lie in the lack of knowledge about it. Scientists who simply use HPLC for their needs are accustomed to reversed phase HPLC, which is a simple technique in relation to HILIC.
To develop an appropriate HILIC separation I would advise sticking to the following rules:
1) In RP mode a stationary phase needs very little equilibration. On the contrary, in HILIC mode any new packing should be firstly saturated with water by washing it with AcN-water 1:1, and then it should be left in MP overnight, and then conditioned by fresh MP for several hours.
2) There is no book or HPLC guideline that would explain how to choose the right stationary phase (SP) for HILIC.
The critical parameter here is pore size distribution; in HILIC mode it has the dramatic effect on plate count, peak shape and retention time repeatability. The one should choose for HILIC a silica-based packing with the narrowest pore size distribution, but (alas!) this parameter is not specified by any HPLC column producer.
Please, take a look at the figure. You see the isocratic HILIC/IC separation of major water-soluble vitamins on IBSnutri BV-1.1. This specialty HPLC column provides excellent peak shapes for analytes and minimal equilibration time precisely because of narrow pore size of intact silica.
From my observation, many HPLC R&D specialists have a habit to disregard what happens in the elution window covering the region from start to void time. Ok, they say, from time to time I can definitely see some signals there, so what?
Meanwhile, all signals in that region are also chromatographic peaks that can be also used for quantification of target compounds. Compounds that elute before void time are excluded from the stationary phase; thus, the corresponding HPLC modes are called exclusion modes. Low molecular weight pharmaceuticals which cannot be excluded by size, can be excluded by their charge only. The corresponding LC mode is called ion exclusion mode, or IEX mode.
It is understood that the specificity of an IEX separation should be carefully studied and verified for all types of real samples to be analyzed. But generally, nothing prevents R&D HPLC specialists from using IEX in their practice.
Please, take a look at the figure. You see the isocratic HILIC/IC/IEX separation of ‘Pentalgin’ pharmaceutical preparation major compounds on IBSpharm HS-1. This specialty HPLC column provides separation of the acidic compound, naproxen, due to IEX mechanism. The void time on this chromatogram is located between naproxen and acetaminophen peaks.
The conclusion is that IEX chromatography can be used for the wide range of pharmaceutical applications.
#5 Why not ion exclusion: analyzing pharmaceutical combinations
#6 Drug screening: group selective HPLC stationary phases are the choice
HPLC separation can be considered as group selective if the elution windows for analytes belonging to different chemical classes do not overlap.
I am a big fan of group selectivity since I’m not an HPLC/MS user, and first of all, I used to work with non-specific detectors like UV/Vis, RID, etc. It means that I always lacked detection specificity, which is the life story of every HPLC specialist accustomed to conventional HPLC detectors. You may have all standards, but if a sample contains too much matrix components… you’re done.
Meanwhile, group selective stationary phases fix this situation. They provide some clear space on a given separation chromatogram that is reserved for your target compounds only. Very good!
Group selective stationary phases also serve the objectives of researchers. They greatly facilitate identification of unknown compounds by providing the possibility of classifying them in a certain chemical class.
Thus, such stationary phases are the best choice for drug screening in drug discovery, forensic, pharmaceutical and biotech applications.
Please, take a look at the figure. You see isocratic separations obtained on IBSpharm HS-1 illustrating group selectivity for antihistamines, i.e. compounds containing one alkyl amine and one pyridine moieties. In turn, the group selectivity for alkyl monoamines is provided by IBSpharm MA-1.
The conclusion is that group selective stationary phase is always a better choice than non-group selective one.