18


May/June 2011


analytes. One benefit of these mobile phases is they are typically quick and easy to prepare (for example they are not often pH adjusted and are used ‘as is’). However, as these additives are organic molecules (in comparison to inorganic buffers such as phosphate or borate), they often have appreciable background absorbance and (in our experience) often contain impurities which can negatively impact chromatographic gradient profile, peak identification and limits of detection. Additionally, these additives are used to simply adjust the pH of the mobile phase and typically offer little buffering capacity. For example, 10 mM ammonium acetate has an aqueous pH of 6.8 which has no buffering capacity. If these mobile phases are used for project progression activities such as SIMs, this may lead to issues with irregular and non-reproducible analyte retention times, poor peak-shapes and ultimately poor method robustness. A list of some organic additives typically used in LC/MS analyses are listed in Table 1.


During method development if lower limits of detection or increased robustness are required, then inorganic additives should be used. Most often, the additive chosen is a phosphate based salt. Phosphate has a wide buffering capacity (pKa


values of = 2.15, 7.20


and 12.30) and excellent UV opacity down to wavelengths as low as 200 nm [11]


. This often


makes phosphate the buffer of choice for method development, particularly in late- stage pharmaceutical development where method transferability is very important and many, if not all, process impurities and degradents are known [e.g. 12,13


Method Column


Mobile Phase Organic Solvent (‘A’)


(‘B’)


1 Waters Acquity 0.1% formic acid acetonitrile Shield RP18


(pH 2.6) 0-1.7 min 5% B


1.7-8.7 min linear to 95% B


8.7-10.4 min 95% B


2 Waters Acquity 10 mM C18


methanol ammonium acetate (pH 6.8)


3 Waters Acquity 0.1% ammonium methanol Phenyl


hydroxide (pH 10.6)


0-1.7 min 5% B 1.7-8.7 min linear to 95% B


8.7-10.4 min 95% B


0-1.7 min 5% B 1.7-8.7 min linear to 95% B


8.7-10.4 min 95% B


Table 2. Organic buffers, solvents and columns investigated in this study.


) framework for SIMs or used alone e.g. for transferring phosphate based methods to MS-friendly buffers for new impurity identification.


If correlations were identified, then this approach could be used as part of a method development strategy whereby a method developed with MS compatible mobile phases for impurity identification could be transferred directly to an appropriate phosphate method for increased UV sensitivity and method robustness. This could be incorporated into a Quality-by-Design (QbD [16]


separations were investigated in an effort to identify alternative mobile phase additives offering higher UV sensitivity and reduced baseline artefacts.


Dihydrogen potassium phosphate and dipotassium hydrogen phosphate (both anhydrous) were purchased from Fluka Analytical (HPLC grade). Phosphoric acid (Fluka Analytical puriss – 85% in water, HPLC grade) was used to pH adjust the mobile phases to the identical pH of the organic buffers they were being compared with.


]. The downside


of utilising phosphate buffers are obviously incompatibility with MS detection, they are more aggressive on silica based columns [14] and also have lower solubility in organic solvents meaning that gradients to high organic fractions are not possible (typically <80% (v/v) solvent depending on organic solvent and salt type, salt concentration and pH).


The primary focus of this investigation was to identify if retention correlations existed between organic buffers and their potassium phosphate alternatives at the same pH, solvent and column types. Analysis at the same pH was important in this work as pH is the dominant contributor to analyte retention for ionisable compounds under reversed- phase conditions [15]


and could lead to


significant deviations in retention and selectivity if not held constant. Correlations were assessed by comparing retention times of the analytes under both sets of conditions and assessing any changes in chromatographic selectivity.


Additionally, high pH buffers were also investigated for SIM development. A comparison of analyte retention in alternative buffers to a commonly utilised generic method employing ammonium hydroxide was examined. Baseline characteristics (such as prevalence of system peaks and effect on peak sensitivity) of the alternative buffers were noted.


Experimental Samples & Reagents


33 compounds were utilised in this study which were families of proprietary Pfizer molecules including the active pharmaceutical ingredients (API), synthetic precursors and process related impurities (PRIs) covering a range of acid, basic, neutral and zwitterionic character. These were typically combined in groups of related compounds at 0.1 mg/mL each in methanol - water (50:50, v/v).


Acetonitrile (Sigma-Aldrich Chromasolv) and methanol (Sigma-Aldrich Chromasolv) were used in this study. Formic acid (HCOOH – Fluka Analytical purissLC-MS grade ampoules) and ammonium acetate (NH4


OOCCH3


grade) were investigated in this study. Additionally, alternatives to ammonium hydroxide (NH4


Purified water was obtained from a Millipore MilliQ Gradient A10 system producing water of 18.2 MΩ.cm and < 3 ppb total organic carbon quality.


The following organic additives and buffers were utilised for the high pH investigation; ammonium formate, acetate and bicarbonate, pyrrolidine, 1-methylpiperidine, triethylamine acetate and the Goods buffers (BioXtra grade) - CAPS, CAPSO and glycine which were all obtained from either Fluka Analytical (puriss LC-MS grade) or Sigma (SigmaUltra grade) except pyrrolidine (Alfa-Aeser). These additives were compared to a mobile phase of 0.1% ammonium hydroxide (unadjusted pH = 10.6). The additives for comparison were prepared at 10 mM (solids) or 0.1% (liquids) concentration and adjusted to a pH of 10.6 with ammonium hydroxide (Fisher) or glacial acetic acid (Sigma Aldrich) as appropriate.


Instruments – Fluka Analytical purissHPLC OH – Fluka Analytical >25% in water ampoules LC-MS grade) for high pH


UPLC experiments were performed on a Waters Acquity UPLC (with PDA detection). The system was controlled through Waters’ Empower 2 software. Both organic mobile phase additive and phosphate experiments were performed on the same system with the same column, thus mitigating any effects from different system dwell volumes or column differences and aging in the correlations. This


Gradient


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52