[Readers Insight] The Starting Point in Method Development

Choosing the starting point for method development has long troubled many analysts in pharmaceutical testing—especially now with the increasing number of novel drugs. When presented with an unknown compound and no reference literature, how should one select the mobile-phase system, column, diluent and other chromatographic conditions? What logic underlies these choices?

In this article, we study and analyze a method recorded in the pharmacopoeia as an example to illustrate the approach. The example entry we use is “Determination of Aspirin Related Substances” in the Chinese Pharmacopoeia, where we examine its method settings, identify potential optimizations, and propose improvements.

Solvent: Methanol containing 1% glacial acetic acid.

Sample solution: Accurately weigh about 0.1 g of the sample into a 10 mL volumetric flask, add an appropriate amount of solvent, shake to dissolve and dilute to the mark, then mix well.

Reference solution: Accurately pipette 1.0 mL of the sample solution into a 200 mL volumetric flask and dilute to the mark with solvent; mix well.

Chromatographic conditions:

• Packing material: C18 bonded silica.
• Mobile phase:
  A) Acetonitrile–tetrahydrofuran–glacial acetic acid–water (20:5:5:70)
  B) Acetonitrile
• Detection wavelength: 276 nm
• Injection volume: 10 μL
• Gradient elution:

System suitability requirements: The retention time of the aspirin peak is approximately 8 minutes; the resolution between the aspirin peak and the salicylic acid peak must meet the specified requirement.

The diluent is methanol containing 1% glacial acetic acid, whereas the initial composition of the mobile phase (A) contains no methanol and comprises 70% aqueous phase. Such a match can produce solvent effects that degrade peak shape. 

Because the injection volume here is relatively small (10 μL), solvent effects may not be evident; however, when selecting a diluent, one should, as far as possible, use a system similar in composition to the initial mobile phase to minimize the risk of solvent effects.

Mobile phase A contains 5% tetrahydrofuran (THF), the purpose of which is unclear. In chromatography, THF is typically used at concentrations <20% and is generally employed to improve separation in complex impurity profiles. From the system suitability requirements, it appears the only impurity of concern is salicylic acid, which differs substantially in hydrophobicity from the main component; separation should not be difficult and, in theory, THF should not be necessary.

From a reagent standpoint, THF has a distinct odor and most commercial THF contains the antioxidant BHT to prevent oxidation; BHT has strong UV absorbance and is therefore unfavorable at low detection wavelengths (in this method the wavelength is 276 nm, so baseline issues may be negligible). \

In addition, THF can swell PEEK materials, and prolonged use can make PEEK brittle and prone to cracking; particulates may form and even block tubing. Therefore, THF should be avoided on instruments containing PEEK components.

The acetic acid content in mobile phase A is 5%, which is relatively high. Acetic acid’s useful buffering range is generally pH 4–6; a 5% acetic acid solution has a pH of about 2.4, outside the buffering range. 

From structural analysis, both the main compound and the impurity contain a benzoic acid moiety: because the carboxyl group is conjugated with the aromatic ring, the benzoic acid derivative exhibits stronger acidity than acetic acid (i.e., a lower pKa). To ensure adequate retention it is desirable to keep these species in their molecular (non-ionized) form; the acetic acid added serves this purpose. 

However, since acetic acid is weaker than the benzoic acid derivative, this may explain why such a relatively large amount (5%) was used.

The gradient run time is 60 minutes, while the main peak elutes at about 8 minutes; the run time is therefore excessively long.

The main compound possesses an additional acetyl group compared with the impurity and is therefore expected to be more hydrophobic; a conventional C18 column should be sufficient for separation. Given the impurity profile (if salicylic acid is the sole significant impurity), a shorter column—50 mm or even 30 mm in length—could be selected to greatly shorten run time per injection.

Use 0.1% aqueous phosphoric acid as Mobile Phase A. 

Phosphoric acid is a moderately strong acid that dissociates in three steps; the first dissociation has an effective buffering range of approximately pH 1.12–3.12. A 0.1% phosphoric acid aqueous solution has a pH near 2 and is fully adequate to suppress ionization of the benzoic acid carboxyl group.

This mobile phase further helps with lowering detector wavelength, see below.

In the presented method the sample concentration is 10 mg/mL, which is relatively high, and the detection wavelength chosen is 276 nm. This wavelength may correspond to a local minimum in the absorbance spectrum and the absolute absorbance may not be high, resulting in poor sensitivity; hence the elevated sample concentration to compensate. 

Therefore, it is recommended to employ an aqueous phosphoric acid–acetonitrile mobile phase, whose mobile-phase UV cutoff is <210 nm (in contrast, a THF system that contains BHT typically requires a detection wavelength above ~260 nm.), allowing improvement to sensitivity while also lowering detection wavelength and sample concentration.