17


Correlation of Analyte Retention in Organic and Inorganic Mobile Phases to aid Liquid Chromatography Method Development


Paul Ferguson1 * and Ronan Huet2


1Research Analytics, Pfizer Global Research & Development, Sandwich, Kent, CT13 9NJ. *Corresponding author: paul.ferguson@pfizer.com 2Devices Centre of Emphasis, Pfizer Global Research & Development, Sandwich, Kent, CT13 9NJ.


Many liquid chromatography methods are developed using organic mobile phase additives which allow compatibility with mass-spectrometric (MS) detection. However, these types of additives often give high UV absorbance which can lead to low level impurity quantitation issues. Additionally, these additives often have little or no buffering capacity at the pH they are typically used, which in turn can lead to variability in analyte retention time. A rational approach for the selection of phosphate buffers from organic based mobile phase additives of the same pH (acidic or neutral) in liquid chromatography stability indicating method (SIM) development may provide a solution to this problem. Excellent correlation was observed for analyte retention (33 test analytes) in switching from an organic based mobile phase additive to an appropriate potassium phosphate buffer at low and mid pH. This approach provides a basis for developing SIM methods under mass-spectrometer friendly conditions and converting them directly to phosphate methods (or vice-versa) which typically provide higher UV sensitivity and retention robustness while maintaining the elution order and chromatographic resolution observed with the organic mobile phase additives.


Keywords: UHPLC, phosphate buffers, retention correlation, ion-pairing Introduction


In the pharmaceutical industry, early clinical phase analytical method development requires a balance between spending sufficient time and effort in developing a fit- for-purpose method against the very real possibility that the compound may be halted before the next project milestone is reached (e.g. due to toxicology or compound absorbance issues). Accordingly, the starting point for chromatographic method development typically involves screening relevant samples through a set of generic methods and choosing the conditions offering best retention, analyte resolution and peak shape [1-4]


. The method providing best global


resolution is generally selected and further optimisation undertaken. This process is often supplemented using other key information (both measured and predicted in- silico e.g.physical-chemical parameters such as pKa


and/or Log D) and chromatographic predictive software such as DrylabTM (Molnar Instiut, Berlin, Germany) [5,6], ChromSwordTM


(Software Entwicklung, Muehltal, Germany) [7] or LC SimulatorTM Canada)[8]


(ACDLabs, Toronto, . This approach often greatly


reduces both the amount of resource and time required to develop a suitable method.


10.6 10.3 - 12.3


0.1% ammonium hydroxide (NH4


OH) pyrrolidine 11.3 [9,10]


Table 1. Some common mobile phase additives employed in LC/MS work. Note – higher pH mobile phases require suitably stable columns such as Agilent Zorbax Extend, Phenomenex Gemini or Waters BEH/Acquity phases.


3.8 – 5.8 8.2 – 10.2


2.8 – 4.8 8.2 – 10.2


9.7 – 11.7 ammonium (acetate) ammonium (formate) Triethylamine (TEA) (acetate)


The generic screening systems may be based on HPLC or UHPLC instrumentation, but are typically hyphenated to both UV and mass spectrometric (MS) detectors. The use of MS detection, particularly in early-phase pharmaceutical development, is essential for early characterisation of new and unknown


Effective pH range or


commonly used pH 1.9


2.8 – 4.8 3.8 – 5.8 6.8 – 11.3


impurities, while UV detection (often diode- array detection (DAD), also known as photo diode array (PDA)) is typically used for quantitation of impurities by area normalisation. The use of MS detection requires LC mobile phases which are both volatile and promote ionisation of the sample


Additive


0.1% trifluoroacetic acid (TFA) formic acid acetic acid


ammonium bicarbonate pKa <1.0


3.8 (HCOO- 4.8 (CH3


) ) ) )


7.8 (H2CO32- 9.2 (NH3+


10.3 (HCO3-


COO- )


)


4.8 (CH3COO- 9.2 (NH3+


3.8 (HCOO- 9.2 (NH3+


) )


4.8 (CH3COO- 10.7 (TEA+


9.2 (NH3+ ) ) ) )


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