Peak homogeneity

The limits of lifetime detection and resolution in on-the-flight fluorescence lifetime detection in hplc were evaluated for simple, binary systems of polycycHc hydrocarbons (70). Peak homogeneity owing to coelution was clearly indicated for two compounds having fluorescence lifetime ratios as small as 1.2 and the individual peaks could be recovered using predeterrnined lifetimes of the compounds. Limits of lifetime detection were deterrnined to be 6 and 0.3 pmol for benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. 

Keller, H. R., Kiechle, P, Erni, F., Massart, D. L., and Excoffier, J. L., Assessment of peak homogeneity in liquid chromatography using multivariate chemo-metric techniques, /. Chromatogr. A, 641, 1, 1993. 

Table 7.53 shows the main characteristics of LC-PB-MS. Of all LC-MS interface methods, LC-PB-MS comes closest to GC-MS (Scheme 7.7). The particle beam is an acceptable choice in cases where sensitivity, volatility and analyte polarity are not an issue. Usually, the function of UV is added to LC-PB-MS this allows the investigation of peak homogeneity. Drawbacks of LC-PB-MS are the low sensitivity and the nonlinearity... 

Peak purity (peak homogeneity) of all relevant peaks, freedom from matrix interferences Peak shapes and efficiencies... 

Investigation of peak homogeneity is an important topic in the validation of the analytical methodology. Method validation requirements are described in the ICH guidelines [1]. Method development involves the separation of drug... 

Chan HK, Carr GP. Evaluation of a photodiode array detector for the verification of peak homogeneity in high-performance liquid-chromatography. Journal of Pharmaceutical and Biomedical Analysis 8, 271-277, 1990. 

When authentic samples of impurities are not available. A stressed drug product can be analyzed to show separation of the most significant related substances. In addition, the peak homogeneity of the stressed sample should be investigated by PDA or mass spectrometry. Alternatively, one may use an orthogonal procedure to verify the method specificity. The orthogonal... 

Marr, D., Horvath, P., Clark, B. I., Fell, A. F. Assessment of peak homogeneity in HPLC by computer-aided photodiode-array detection, Anal Proceed 23 254-257 (1986). 

Degradation product(s) co-eluting with the parent compound in the parent compound assay. Check for peak homogeneity via PDA-UV, LC MS, and/or use an orthogonal separation technique, as discussed earlier. Modify conditions to separate if necessary. 

G. T. Carter, R.E. Schiesswohl, H. Burke, and R. Young, Peak homogeneity determination for the validation of high-performance liquid chromatography assay methods, J. Pharm. Sci., 77 317 (1982). 

A more popular approach for detemining peak purity has been the use of diode-array detectors which were first introduced in 1982 [50]. Peak homogeneity of similiar benzodiazepines [51] and theophylline have been determined by this technique [52]. Rapid-scan detectors are also useful in confirming the presence of known components such as colorants, which are commonly used in drug products [53,54] and identification of related substances [55,56]. 

Another feature of the photodiode array detector is the ability to determine peak homogeneity. To determine the homogeneity of a peak, software algorithms are used to compare the spectrum of the compound at the peak apex with spectra obtained at other points across the peak. In general, peak homogeneity translates into peak purity. However, if two symmetrical peaks are perfectly overlayed as illustrated in Figure 7.5, the peak, while perfectly homogeneous, is impure. 

An example of the use of photodiode array detectors for determining peak homogeneity is in the analysis of /3-carotene,8 where an impurity in the all-trans isomer was positively identified as the cis isomer.9 Since the U.S. Federal Register began to require listing of the presence and concentration of various vitamins on the labels of foods, there has been increased activity in the measurement of these vitamins. /3-Carotene is one such vitamin, and research links the presence of /3-carotene to a reduction in the occurrence of some degenerative deseases such as cardiovascular diseases and cancer. 

Final structural proof of Hez-PBAN came irom demonstration of identical properties of both the isolated native and synthetic Hez-PBAN disulfoxides. Synthetic Hez-PBAN was oxidized to its disulfoxide by the method of Wagner and Fraser (9) Both native Hez-PBAN and synthetic Hez-PBAN disulfoxide demonstrated identical retention times. In addition, the elution profile of a mfacture of equal amounts of native and synthetic Hez-PBAN disulfoxides displayed one peak under the conditions of RP-HPLC Step D. Superimposition of its normalized upslope, apex, and downslope UV spectra indicated peak homogeneity. 

The scope of ultraviolet and visible spectrophotometry can be further extended when combined with a chromatographic separation step such as HPLC. The development of rapid-scanning detectors based on the linear photodiode array permits spectra to be acquired during the elution of peaks. Computer-aided manipulation of these spectra has led to new strategies for the examination of chromatographic peak homogeneity, based on classical techniques in spectroscopy. The use of microcomputers enables the development of archive retrieval methods for spectral characterisation (A. F. Fell etal, J. Chromat., 1984, 316, 423-440). 

Figure 8-14 Determination of peak homogeneity Diode array detection (DAD). (Reprinted from reference 10, with permission.)...

Once the probe gradient is run, check the diode array purity and if LC-MS is available, run as well to check for peak homogeneity. If you have any known precursors or impurities, run them as well to ensure resolution from the main component and to make sure they are adequately retained. The main analyte should elute between k 2-5. If the main component elutes at A = 2-5 and is spectrally pure and the impurities all elute k > 1, the method is complete. If the retention factor of the impurities is below 1, then an isocratic hold at the initial organic composition should be implemented until the minor component (impurity) elutes k > 1 and then a linear gradient can be implemented. The method could be further optimized by increasing the flow rate as long as the backpressure limitation of the system has not been reached. A general rule of thumb is that the backpressure should not exceed 85% of the maximum backpressure for a particular HPLC system. 

Figure 8-41. Determination of peak homogeneity. Chromatographic conditions Column Luna C8(2) 150 x 4.6 mm. MobUe phase 10 mM NLLOAc, pH 5.8, acetonitrile. Column temperature, 40°C flow, 1.0 mL/min injection volume, 10 pL. Linear gradient from 5% acetonitrile to 95% acetonitrile over 20 min with 3-min hold at 95% acetonitrile.

Hyphenated techniques utilizing HPLC plays very important role in drug development process, especially LC-MS. The power of LC combined with an universal detector is an optimal combination to determine related substances and peak homogeneity. Selected articles are referenced for the reader [30, 49,50]. 

Peak Purity Investigations. In order to be able to detect the coelution of unknown substances, peak homogeneity (also termed peak purity) investigations should be performed. 

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