1. 3M Drug Delivery Systems 3 Maggi G. Tebrake a Monica Dolci b, and Roger M. Smith b a) 3M Healthcare Limited, Morley Street, Loughborough, Leics LE11 1EP UK b) Department of Chemistry, Loughborough University, Leics LE11 3TU UK Text Here – Body copy will vary in size depending on how much text you have to fill the whole poster NOTE* - You cannot reduce your Body Copy smaller than 12 pt type on this template. 3M Drug Delivery Systems SCREENING OF LC METHODS FOR THE SEPARATION OF SURFACTANTS EMPLOYED AS PHARMACEUTICAL EXCIPIENTS Reversed-phase liquid chromatography (RP-HPLC) allowing separation based on hydrophobic interactions. Normal-phase (NP-HPLC) and hydrophilic interaction chromatography (HILIC) allowing separations based on polarity. Liquid chromatography at critical conditions (LCCC) was investigated in each chromatographic mode. LCCC allows analytes to be separated solely according to functionality, the chain length does not contribute to the retention. The active parameters considered for the screening were: Temperature Mobile phase composition Organic modifier Stationary phase materials Mass spectrometry (MS) confirmation was employed. The aim of the present study was to evaluate a number of different chromatographic systems for the characterisation of polylactic acids (PLAs). A family of excipients based on PLAs (Fig 1) are employed to improve the pharmaceutical performance of pressurised metered dose inhalers (pMDI) Fig 1: General structure of polylactic acid excipient Separation of PLAs and their potential process impurities (e.g. cyclic PLAs) and degradation products is required to characterise the excipient material. The separation and identification of PLAs can be be challenging. They are not one chemical entity, rather a mixture of molecules of similar chemical properties but varying numbers of repeat units. A number of separation modes were investigated. Compatible orthogonal separations could in future be coupled to achieve comprehensive separation of PLAs and related substances. Introduction Methods Instrumentation The following systems were employed: Agilent HP 1100 LC-MSD Agilent HP 1100 LC-ELSD HP 1090 LC-UV Thermo Finnigan LTQ LC-MSD RP-HPLC Figure 2 shows a PLA sample separated on Spherisorb C6, water/ MeCN mobile phase, linear gradient. Each peak contains individual oligomers Oligomer A Oligomer B Oligomer C Oligomer A Oligomer B Oligomer C Fig 2: Left : Total Ion Chromatogram (TIC) of PLA excipient. Right : Extracted Ion Chromatograms (EICs) of oligomers A, B and C, showing the 3 oligomers eluting in order of chain length (oligomer A being the shortest and C being the longest of the 3) MeCN did not provide the selectivity necessary to achieve separation between cyclic impurities and PLA (Fig 3). RT:0.00 - 35.00SM:15B 0246810121416182022242628303234 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 100 Relative Abundance RT:0.00 - 35.00SM:15B 0246810121416182022242628303234 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 100 Relative Abundance 60 65 70 75 80 85 90 95 31.10 30.97 31.39 30.64 31.46 31.57 30.53 30.37 30.13 29.88 29.68 6.27 29.37 6.63 29.12 7.40 23.70 22.83 28.80 7.84 26.73 5.95 22.51 32.24 8.24 20.41 8.63 19.22 18.34 33.13 16.74 8.98 14.97 13.64 9.43 11.85 5.47 4.94 0.233.821.49 NL: 4.33E5 Base Peak MS MD070207 _42ppmcyc _RPLC Cyclic 60 65 70 75 80 85 90 95 7.87 7.55 8.27 7.18 8.66 6.82 8.98 9.39 6.46 9.73 10.14 10.58 6.12 13.31 14.37 11.03 14.47 11.40 15.42 15.91 5.79 16.17 17.11 17.80 32.49 4.94 4.72 18.74 32.95 19.82 21.96 23.34 2.940.83 24.8931.19 2.3126.51 30.94 NL: 6.85E6 Base Peak MS MD280207 _1082ppm OLA_RPLC PLA Cyclic oligomers PLA oligomers Fig 3: PLA and cyclic impurities, eluting on C4, mobile phase water/MeCN. Left: TIC of PLA and cyclic impurities; Right : EICs of PLA and cyclic impurities and relative EICs When MeOH was used as organic modifier the separation selectivity changed. Resolution between PLA and their cyclic impurities was achieved (Fig 4). Oligomeric separation was also obtained. Cyclic oligomers PLA oligomers Cyclic PLA oligomers Fig 4: PLA sample spiked with cyclic, separation on C4, mobile phase water/MeOH. Left:: TIC of PLA and cyclic impurities; Right: EICs of PLA oligomers and cyclic oligomers. LCCC methods were developed according to Cools et al. [1]. Using C6 stationary phase and MeCN as organic modifier, PLA oligomers co-eluted at 74% MeCN at RT 5.6 min (Fig 5, left). However, LCCC did not provide resolution of PLAs and their cyclic impurities. The cyclic impurities showed a retention time of 5.5 minutes (Fig 5, right). Fig 5: Left : EICs of PLA oligomers D, E and F; LCCC gives RT of 5.6 min for all oligomers. Right : EICs of cyclic oligomers G, H and I; LCCC gives RT of 5.5 min for all oligomers. NP-HPLC/HILIC LCCC on NH2 and CN sorbents, led to separation of PLA and their cyclic impurities. Separation was achieved employing both, aqueous and non-aqueous solvent systems (Fig 6). Fig 6: Left : Overlaid TICs, Spherisorb amino column, 10% n-hexane/90% MeCN, 30 °C Right : Overlaid TICs, Zorbax cyano column 20% Ammonium formate/80%MeCN, 30 °C Spherisorb amino sorbent exhibited HILIC behaviour employing aqueous/MeCN eluent. Zorbax cyano material provided HILIC behaviour at low aqueous content and RP- HPLC behaviour above ~ 20% aqueous in the mobile phase (Fig 7, left). Using non-aqueous/MeCN eluent, neither amino nor cyano material provided a trend in retentions (Fig 7, right). Fig 7: Left : HILIC screening summary, PLA and cyclic impurities, aqueous/MeCN mobile phase Right : NPLC screening summary, PLA and cyclic impurities, non aqueous mobile phase Conclusions In reversed phase mode the retention windows for main excipients and their cyclic impurities overlapped when using MeCN as organic modifier in the mobile phase. However, using MeOH a change in selectivity was observed. Full separation of PLA excipients and their cyclic impurities was achieved. The CN and NH 2 columns showed good separation in both, NP-HPLC and HILIC, at critical conditions. Separation was achieved using differences in the molecule’s functionalities, leading to resolution between the PLA excipients and their impurities. All screening studies were performed qualitatively. The validation of limits of quantification and detection will follow this screening study. Furthermore, the RSDs for excipient and impurity quantification will be measured. The method will be employed for purity analysis of bulk material and stability studies of PLA containing formulations. Literature [1] P.J.C.H. Cools, A.M. van Herk, A.L. German and W.J. Staal, J. Liq. Chromatogr., 17 (1994) 3133.