This instalment of "LC Troubleshooting" examines an issue associated with a liquid chromatography (LC) method for a pharmaceutical
product. Because the method is proprietary, some of the details will be left out of the method description presented here.
This method had performance requirements that were typical for such methods — the average assay value of several replicate
injections had to fall within 98–102% of the expected value. The method range was 70–130% of the nominal value for the drug
in the product. The product contained two active ingredients, which will be referred to as compound A and compound B for the
present discussion. The issues encountered with this method provide a good example for the application of general troubleshooting
principles.
The Method
The method used a 150 mm × 4.6 mm reversed-phase column packed with 5 μm diameter particles and maintained at 30 °C with a
column oven. The mobile phase was 90:10 phosphate buffer–acetonitrile with a flow-rate of 2 mL/min. UV detection was used.
Table 1: Typical results at 130% of the nominal concentration.
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The method had been validated several years ago and was transferred to a second laboratory recently. When adjustments were
made to the method for a new application, the performance requirements for compound B were easily met. Whereas, compound A
passed at concentrations of 100% of the nominal value and below, but it did not meet the ±5% specification window at 130%
concentration (95%). It was found that reducing the injection volume from 20 μL to 10 μL allowed the 130% concentration to
meet the requirements for both compounds, so the method was revalidated for 10 μL injections. Typical results at the 130%
level are summarized in Table 1.
Are the Results Reasonable?
One of the first steps to take when examining any problem related to the results of an LC method is to determine if the data
obtained are reasonable for the method conditions. Three parameters that are examined easily are retention time, peak width,
and peak shape. Let's look at each of these in turn.
Retention: Rather than look at retention times alone, it usually is more informative if we check the retention factor, k. For the best chromatographic performance, it generally is desirable to have k-values between 2 and 10, although most workers will settle for values of 1–20 if necessary. The retention factor is calculated
as
where tR and t0 are the retention time and the column dead time, respectively. The column dead time can be measured by injection of an unretained
compound, such as uracil, but for many purposes, an estimate is good enough. An estimate of t0 for 4.6 mm i.d. columns is
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