Monographs and general chapters that provide public quality standards for drugs, excipients, and dietary supplements are published in the United States Pharmacopeia and the National Formulary (USP–NF)1. About a decade ago, the USP started a global initiative to modernize many of the existing monographs and since then has been actively seeking industry collaborators to assist in the development of such monographs2. The direct participation of the pharmaceutical industry, and other interested stakeholders, in this program is still highly encouraged to assist in providing updated public standards to strengthen the protection of public health. The USP intends to modernize monographs as soon as possible; either by traditional submission from a stakeholder or from USP’s internal laboratory efforts.
Providers of instrumentation and consumables support this initiative through continuous development of products, tightening of specifications, and commercialization of new technical innovations and improvements. For chromatographic analysis, and especially HPLC testing, several new column chemistries (i.e. packings, L classifications, or stationary phases) bound to different base materials are increasingly being developed and introduced. One of the more recent contributions from Merck KGaA, Darmstadt Germany, to support USP endorsed chromatographic quality control, is the SeQuant ZIC-pHILIC methacrylate material, a sulfobetaine type stationary phase graft-polymerized to totally or superficially porous hydrophilic polymer particles. This material has been provisionally assigned the new column classification L1223. The material description is comparable with USP L1144, in the sense that both packings have densely bonded zwitterionic groups with a 1:1 charge balance, but where the L114 material is silica based, rather than a polymer core.
L122, a New Column Classification
The L122 packing has been developed and designed for use in the separation and analysis of polar and hydrophilic molecules and their related substances and applied to hydrophilic interaction liquid chromatographic (HILIC) mode. Development of separations in HILIC mode is analogous to traditional Reversed Phase (RP), in the sense that the eluents are similar, and the main retention mechanism is the same. In both modes the retention is mainly a result of partitioning between the stationary phase and the bulk eluent. The difference is the effect from the hydrophilic portion in the mobile phase and its influence on separation. In HILIC mode, water is the strongest solvent, and to increase analyte retention, the organic portion of the mobile phase needs to be increased, and the water or buffer portions decreased. This will increase hydrophilic partitioning into a water-enriched layer on the stationary phase, and thus increase the retention of the analyte. One easy rule of thumb to remember is that in HILIC most changes will have the opposite effect on retention compared to reversed phase. HILIC stationary phases will also to some extent have ionic interactions with charged compounds. This is true also for nominally neutral stationary phases. Therefore, all HILIC columns should be used with an eluent with some ionic strength: high ionic strength for charged columns like bare silica and amine columns and low for uncharged or zwitterionic stationary phases like L114 and L122. The zwitterionic character and the 1:1 balanced charge, give further possibilities for selectivity by weak electrostatic interactions between the stationary phase and the molecules that are separated. This interaction can be tuned by changing buffer type and concentration, typically in the range 5-50 mM. Buffer pH is also an important parameter to control retention, but also here the thinking is opposite to that of RP mode; more ionized compounds will be more hydrophilic and thus have more retention on a L122 packing.
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Choline Citrate: A New Method
Packings L114 and L122 can be useful tools in developing methods for molecules in the USP monograph modernization list, where in fact many of the molecules are of hydrophilic character. Additionally, many current official USP Monographs for hydrophilic compounds are still based on wet chemistry methods and lack chromatographic assay or related compound methods. L122 has now been proposed to be used for the “content of Choline test in the monograph for Choline Citrate”5. The new method uses a 150x4.6 mm L122 column with a charged aerosol detector (CAD), and where the relative retention times for choline related compound A, choline, and citrate should be 0.86, 1.00, and 2.83, respectively. The chromatographic resolution should not be less than (NLT) 2.0 between the choline related compound A and the choline peaks.
Traditionally the analysis of choline salts utilizes HPLC-UV instrumentation and a derivatization reaction. The derivatization procedure provides more selectivity, through the addition of chromophores to the choline salt skeleton. However, this approach suffers from interferences with typical counter ions such as tartrate or citrate and known impurities do not react with the derivatization agent. A charged aerosol detector (CAD) is a universal detector with enough sensitivity to detect nonvolatile analytes, their impurities, and related compounds. HPLC-CAD instruments are especially well suited for analytes that are salts, whose counter ions also can be detected, identified, and quantitated.
The combination of HILIC-CAD, using the L122 packing, solves the analytical need and provides a solution that improves pharmaceutical quality control testing.
References:
- USP, 2020, USP-NF, USP, Viewed March 23, 2020, <https://www.uspnf.com/>
- USP, 2019, Monograph Modernization History, USP, Viewed March 23, 2020, <https://www.usp.org/get-involved/partner/monograph-modernization-history>
- USP, 2020, L122, USP-PF, viewed March 23, 2020, <https://online.usppf.com/usppf/document/GUID-5F5A3673-A11D-470B-B4A3-9B3343696E0A_10101_en-US> (require log-in, free-of-charge registering)
- USP, 2019, L114, USP-PF, viewed March 23, 2020, <https://online.usppf.com/usppf/document/GUID-373BD1D0-801C-438F-80CC-323DCAAB6D67_10101_en-US?highlight=L114> (require log-in, free-of-charge registering)
- USP, 2020, Choline Citrate, USP-PF, viewed March 23, 2020, <https://online.usppf.com/usppf/document/GUID-45240245-4A63-4C65-8CDA-1F35BF4E78AB_10101_en-US> (require log-in, free-of-charge registering)