Peak purity detection using

FIGURE 17 Peak purity detection using (a) LC/DAD-UV vs. (b) LC/MS (courtesy of Waters Corp.). 

Cameron G, Jackson PE, Gorenstein MV (1993) A new approach to peak purity assessment using photodiode array detection. Chem Aus, 288-289. 

Peak purity tests are used to demonstrate that an observed chromatographic peak is attributable to a single component. Mass spectrometry is the most sensitive and accurate technique to use for peak purity evaluation because of the specific information derived from the analysis. However, a good number of HPLC methods use mobile phase conditions that are incompatible with mass spectrometry detection. In this case, PDA spectrophotometers using peak purity algorithms may be used to support the specificity of the method. Almost all commercially available diode array detectors are equipped with proprietary software that will perform these calculations. Although this technique is more universal in application to HPLC methods, the data provided is neither particularly... 

The validation of the peak purity is important for selectivity. The efficiency should be optimized to avoid co-elution of different analytes. Two-dimensional detection is a quick and convenient method to check peak purity. CE/UV coupling is most common,and instruments are commercially available. ° The possibilities of CE/MS have already been discussed. The use of several separation systems with different selectivity is a more time-consuming method to test peak purity. 

Caution needs to be exercised in confirmation of method specificity, as peak purity cannot be inferred from the absence of any mass spectrometric signals, as an absence could be because of ion suppression, or the response may too low to be detected, or the unknown may have different ionisation properties. Peak purity should be tested by using an orthogonal technique. 

Time-shcing is a variation of the stop-flow mode where the flow is stopped in a time-dependent manner (e.g. every 15 s) for the whole chromatographic run, in a sense the ultimate approach in slow-flow. When the flow is stopped, sufficient scans can be made to give the desired level of detection before moving on to the next time-slice. The whole process can be automated through software control. There is no requirement for an UV detector, and the approach has been used to identify non-UV-active components in drug substances [48]. The peak purity of... 

Photodiode array detection has three major advantages for HPLC analysis (26) (a) multiple-wavelength detection, (b) peak identification, and (c) peak-purity determination. Since PDA can record the characteristic UV spectra of the different phenolics as they elute from the column, characterization and peak-purity information can be facilitated through comparison of the spectra at the front, the apex, and the tail of each peak. Furthermore, the rapid calculation of absorbance ratios between different wavelengths is possible, which can be used to classify the spectra by functional groups or by other criteria (Table 1). 

U.K.) was used with solvent flow rate of 6 ml/min. With the recorder set at 0.2 absorbance units full scale, the respective detection wavelengths and sample loadings were 450 nm (50a 1 and 20/ 1) and 260 nm (2/ 1). Assessment of peak purity using 260 nm takes advantage of the increased sensitivity and also detects colorless but UV-absorbing compounds. 

Fig. 3 Investigation of chromatographic peak purity by diode array detection (A) spectra of drug substance and impurity (B) coelution of a mixture containing about 10% impurity (C) coelution of a mixture containing about 0.5% impurity. The spectra were obtained in the peak maximum, at about 5% and 50% of each side of the peak. The normalization was performed with respect to the first spectrum at the peak front (a matchfactor of 1000 means identical spectra) using commercial software.

A Thermoseparation Products Co. HPLC was utilized, consisting of the following components Constametric 3500 pump, AS 3000 autosampler, and UV 1000 detector. A Zorbax XDB Cig (5 pm, 4.6 x 150 mm Agilent Technologies) column was used. The mobile phase flow rate was I mL/min and the column oven temperature was maintained at 40°C the analytes were detected at a wavelength of 226 nm. The injection volume was 25 pL. Data acquisition was performed with the software, PC 1000. Peak purity was checked with a photodiode array detector (UV6000 LP Thermoseparation Products Co.). 

Peak purity analysis software allows users to sample spectra at equidistant points across an HPLC peak. In general, the poorer the resolution between potentially coeluting peaks is, the more desirable it is to use greater numbers of data points to detect the impurity. Traditionally, spectra have been sampled upslope, at the apex, and downslope of the eluted peak. This selection pattern may overlook the presence of impurities near the peak extremities. However, acquisition of many spectra may increase calculation and display time without adding significant information. 

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