Gradient impurity testing

Figure 5.13. A good vs. a bad blank chromatogram from a gradient trace analysis for impurity testing of pharmaceuticals. The ghost peaks from the blank injection are derived mostly from the trace contaminants in the weaker mobile phase, which are concentrated during column equilibration. Reprint with permission from reference 16.

Figure 6.7. An example of impurities testing using gradient HPLC of a pharmaceutical product stored under accelerated stability conditions, noting the presence of excipient, preservative (butylated hydroxytoluene, BHT), impurities (Imp), and degradants (DG). HPLC conditions column Waters XTerra MS18, 150 x 3mm i.d., 3pm mobile phase (A) 16mM ammonium bicarbonate, pH 9.1 (B) acetonitrile gradient 3% B to 45% B in 25min flow rate 0.8mL/min at 50°C detection 280nm. Additional information can be obtained from reference 22.

Figure 6.11. An example of a composite assay (combining both assay and impurity testing in one method) for a drug substance in early development. Note that the absorbance of the API must be <1.5 absorbance units (AU) to prevent detector saturation. Since the method has some deficiencies (e.g., partial resolution between several peaks), an improved gradient method was thus developed (Chapter 8).

Trends toward MS-compatible, gradient methods for impurity testing... 

Figure 10.15. Chromatograms illustrating gradient shift problems at 230 nm encountered during method development of an impurity test method for a drug substance (Section 8.8.2). The UV spectrum of 0.05% TFA vs. water is shown in the inset, showing considerable absorbance at...

In general, it is fair to state that one of the major difficulties in interpreting, and consequently in establishing definitive tests of, corrosion phenomena in fused metal or salt environments is the large influence of very small, and therefore not easily controlled, variations in solubility, impurity concentration, temperature gradient, etc. . For example, the solubility of iron in liquid mercury is of the order of 5 x 10 at 649°C, and static tests show iron and steel to be practically unaltered by exposure to mercury. Nevertheless, in mercury boiler service, severe operating difficulties were encountered owing to the mass transfer of iron from the hot to the cold portions of the unit. Another minute variation was found substantially to alleviate the problem the presence of 10 ppm of titanium in the mercury reduced the rate of attack to an inappreciable value at 650°C as little as 1 ppm of titanium was similarly effective at 454°C . 

The use of the laser-light-scattering detector permits the record of excellent chromatograms in gradient elution. An analysis of the test mixture of standards is shown in Fig. 37B for comparison with the same analysis obtained in isocratic conditions. The analysis lasts about 30 min before the elution of BBB instead of 18 min in isocratic conditions, but the first part of the chromatogram is considerably improved, and a number of impurities in the standards can be resolved. 

Fig. 37 Chromatograms of a test mixture of homogeneous tryglyceride standards. Columns 250 X 4-mm ID LICHROSPHERERP18 (5 /rm) constant flow rate l.Oml/min. A, isocratic conditions solvent chloro-form/acetonitrile (49 51 v/v) (one column). B, gradient elution chloroform in acetonitrile. Starting conditions 30 70, program rate 1%/min for 25 min to 55 45 and then 3%/min for 15 min to 100 0 and hold at 100% chloroform for 8 min (two columns in series). Some impurities are contained in the standard compounds.

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

In order to identify any excipient- and packaging-related impurities in the formulation, placebo cores and hlm-coated placebos, were prepared using the same excipients as in the active tablets. The placebo cores and film-coated placebos, as well as active cores and hlm-coated active tablets were set up on a stability study in high density polyethylene (HDPE) bottles with and without desiccant and in foil-foil blisters. At the designated time intervals, the tablets were tested for purity by gradient HPLC analysis. 

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