How Isocratic HPLC Solved a Pharmaceutical QA Problem

In the interest of better analytical selectivity in the quality control of an intermediate pharmaceutical product, we switched from a GC methodology to isocratic HPLC. Operating in a GLP­regulated environment, we also regarded the validation features incorporated in the HP 1100 Series chromatography system an essential asset for validating the analysis and verifying proper instrument function. GC: Byproducts Not Resolved We were aware that the compound of our interest (for proprietary reasons, we cannot identify its components by name) contains two known byproducts in small concentrations. The chromatogram in Figure 1 reveals that the GC column we used could not resolve the main component from the impurities. Peak 1 (at 1.05 min) is known to be the solvent peak, Peak 2 is an added internal standard, and Peak 3 is the main component. The FID failed to detect the two impurity peaks. HPLC: Better Reproducibility Eventually, we turned to HPLC with its various mobile and stationary phases and settled on a simple isocratic separation as the method of choice. The chromatogram in Figure 2 clearly shows the two baseline­separated byproducts preceding the main peak. In contrast to the customary GC approach, the high injection reproducibility of today's HPLC instrumention even made the addition of an internal standard completely unnecessary. We used an HP 1100 Series HPLC system consisting of an isocratic pump, an autosampler, a thermostatted column compartment, a variable­wavelength UV detector, and the ChemStation software for system control and automated data evaluation and validation. This system was also used for the development and validation of an isocratic HPLC method for still another product, a color additive for alimental products. Summarizing the Benefits The rationale for choosing the HP 1100 HPLC system for both regulated analyses can be summarized in terms of clear benefits: A single system configuration for both applications Easy programming of the numerous functions of each module Time­programmable operation sequences, such as initiating operation of detector lamp and pump to obtain a stable baseline and equilibrated column before the workday begins Excellent reproducibility of retention times (the column packing is silica, known to be sensitive to even slight variation in solvent composition) An injection volume variable from 0.1 to 100 microliters without any hardware modification The option of programming an external needle wash The flexibility of the data analysis Column Conditions Recorded In addition to these benefits, the system offers easy monitoring of column conditions throughout the unit's operating life. All important events are recorded in software logs and a dedicated column identification module; the number of injections, the solvent volumes ­­ all are monitored and listed in individual sample reports. An early maintenance feedback (EMF) informs the user when a predefined limit for lamp usage time has been exceeded. All of these features answer the requirements of Good Laboratory Practice (GLP). Some results of the method validation are detailed in Figures 3, 4, and 5. Measuring Precision and Detection Limit Figure 3 represents the linearity curve, with X the concentration of the pharmaceutical product, and Y the UV detector response. The correlation coefficient is 0.9999. The validation report (Figure 4) demonstrates the precision of this isocratic HPLC methodology, with a typical relative standard deviation of 0.5% for peak areas, calculated from 10 consecutive injections of the same solution of the product. This series of runs was repeated for the scale formulation, with 0.50% RSD. Two additional series of reproducibility measurements for 10 different preparations of powder and scale produced 0.59% and 0.58% RSD, respectively. Tests performed to establish the minimum detection limit (Figure 5) resulted in 6.390E­02 mg/L detection limit (1.5 times detector noise) and 1.118E­01 mg/L quantitation limit. Finally, to compare our original GC methodology to our newly adopted HPLC approach, we analyzed six different lots of the product. RSD for the GC method was 1.83%, and 0.56% for HPLC ­­ a revealing demonstration in favor of the latter.