HPLC-Based Assays

Soverini S, Martinelli G, Amabile M et al. Denaturing-HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to Imatinib. Clin Chem 2004 50 1205-1213. Jaeobberger JW, Sramkoski RM, Frisa PS et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a eell-based measure of BCR-ABL kinase activity. Cytometry A 2003 54 75-88. 

Third, the hazards of handling and disposing of radioisotopes have made it desirable to find alternative methods of assay. When the assay involves fluorescent or intensely absorbing substrates or products, the sensitivity of HPLC-based assays rivals that of radiochemical assays. 

Figure 9.78 HPLC-based assay for sucdnyl-CoA synthetase from rabbit liver mitochondria. One hundred microliters of diluted supemate was added to 3.0 mL containing 50 mAf succinate, 33 mAf Na-Hepes, pH 7.4, 5 mAf MgCl2,1 mAf CoA, 1 mAf GTP, and 5 jug of oligomycin. The reaction was run at 30°C and aliquots of 0.30 mL were removed at 1-minute intervals and transferred to autosampler vials containing 0.2 mL of 0.2 Af formic acid. Nucleotides were separated by HPLC. The UV detector was set to 254 nm. The chromatographic profiles for the reaction after 0, 5, and 10 minutes are shown. Peaks (top profile) 1, GMP 2, GDP 3, GTP 4, CoA and 5, succinyl-CoA. (A) Linearity of succinyl-CoA (S-CoA) formation, expressed as the percentage conversion of CoA to sucdnyl-CoA, with time. (B) Linearity of S-CoA formation with micrograms of protein added for 5 minute assays carried out in a volume of 0.30 mL. The latter assays were carried out in duplicate. The values shown are averages of the duplicate assays. (From Lambeth and Muhonen, 1993.)...

The of the MF + C02/formyl-MF couple is —497 mV and that of HVH2 is —414 mV [184]. Thus, in vitro H2 is not a good electron donor for Reaction (48). However, an HPLC-based assay in the direction of C02-reduction, using Ti(III)-citrate ( 0 = —480 mV) as an artificial electron donor, has been described [185]. In the opposite direction, the enzyme is easily assayed spectrophotometrically using the artificial electron acceptor methylviologen ( = —446 mV) ... 

PNAE was the first enzyme detected in the ajmahne pathway. Detection and prehminary characterization were first reported in 1983. This enzyme turned out to be an extraordinary highly substrate selective protein which has been reported several times. In order to achieve a comprehensive purification of PNAE, an HPLC-based assay was developed for... 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

A procedure to determine PIR residues in bovine milk using HPLC with the derivatization step for UV detection has also been published (211). The PIR was extracted from milk after protein precipitation and a two-step LLE procedure. The extract was evaporated to dryness, dissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (FMOC). The de-rivatized extract was analyzed by reversed-phase HPLC. Overall recovery was 89%, with 4% for coefficient of variation. A linear regression analysis of HPLC/UV results was compared with the HPLC/MS assay (209,210). The procedure takes about 2.5 hours to complete six or eight samples. Pirlimycin is stable in milk frozen to —60°C or below for at least 3 months. 

It should be noted that the tests and specifications listed in Table 7 are provided for guidance and should be set for each individual method based on its application and performance. For example, there may be no resolution requirement for an HPLC dissolution assay. For the same method, it may also be impractical to set the injector repeatability requirement at an RSD of < 1% if the peak response for the API is very small. Again, in the same example it is also impractical to require a check standard to be within 1% of the working standard if the RSD requirement for replicate analyses of the working standard is <2 %. Some ion exchange columns may not provide plate counts of > 2000 or tailing factors of < 2. 

There are several reasons for the development of HPLC-based methods for assaying enzymatic activities. First is the desirability of accurately assessing enzymatic activity in preparations as near the biological state as possible. This often requires the use of turbid preparations, and the product to be measured must be separated from other components of the reaction mixture. However, the use of crude preparations often carries the price of needing to correct for secondary reactions. HPLC allows secondary reactions to also be measured. 

In our laboratory, we have developed a novel MALDI-MS based assay in combination with separation techniques to rapidly identify and confirm the presence of viral proteins (immature or mature) and impurities in the rAd vector [138], The approach combines powerful multidimensional analytical techniques to fully characterize viral proteins, including RP-HPLC, SDS-PAGE, MALDI-MS, MS/MS, and database searching methods (Figure 19-24). This assay involves dissociation/separation of intact viruses by RP-HPLC, separation of the SDS-dissociated viruses by SDS-PAGE, and enzymatic digestion of the dissociated viral proteins from the RP-HPLC fractions and the gel bands, followed by MALDI-MS, MALDLpost source decay (PSD) studies, and database search. 

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