Gradient elution, reversed-phase HPLC based

A clean-up process-scale reverse-phase HPLC (RP-HPLC) step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 801 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 8.7). Separation is achieved using an acidic (often acetic acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30% acetonitrile). 

Two predominant phenolic compounds (neochlorogenic and chlorogenic acids) in prunes and prune juice can be analyzed by reversed-phase HPLC with diode array detection along with other phenolic compounds (65). Phenolic compounds were extracted from prunes with methanol and aqueous 80% methanol and analyzed by HPLC. Ternary-gradient elution (a) 50 mM NaH4H2P04, pH 2.6, (b) 80% acetonitrile/20% (a), and (c) 200 mM phosphoric acid, pH 1.5, was employed for an 80-min run time. Four wavelengths were monitored for quantitation 280 nm for catechins and benzoic acids, 316 nm for hydroxycinnamates, 365 nm for flavonols, and 520 nm for anthocyanins. Phenolic analysis of pitted prune extract is presented in an HPLC chromatogram in Fig. 9, which is based on work done by Donovan and Waterhouse (65). 

In contrast nucleosides are bases of moderate hydrophobicity. They are easily resolved by reversed phase HPLC but again their complexity in biofluids means gradient elution is usually required. 

Gradient reverse phase HPLC with mass spectrometric detection is used to confirm Caribbean or Pacific CTX-1 in the fish tissue extracts. Identification is based on mass and retention time equivalent to C-CTX-1 or P-CTX-1 reference materials. The concentration of reference standard used is 10 ng/mL. The LC/MS/MS system consists of an LC system (Agilent Technologies Model 1100, Palo Alto, CA) coupled to a 4000 Q Trap mass spectrometer (Applied Biosystems, Foster City, CA). LC separations are performed on a Luna C8 (2) column (2.0 x 150 mm, 5 pm, Phenomenex, Torrance, CA) at a column temperature 40°C. Mobile phase is water (A) and acetonitrile (B) in a binary system, with 0.1% formic acid as additive. The elution gradient is 35% B for 2 min, linear gradient to 80% B at 30 min, 95% B at 35 min, hold at 95% for 10 min, return gradient to 35% B at 50 min, and hold for 10 min before the next injection. 

Microanalytical LC-MS methods for the analysis of BR-derived boronates have also been developed, using either atmospheric pressure chemical ionization (APCl) or electrospray ionization (ESI). APCl-based LC-MS has been used to analyze naphthaleneboronate derivatives of BRs in this case, optimal results were obtained by using a reverse-phase HPLC acetonitrile-water gradient to elute a CIS column [67]. Typical ions observed in the positive-ion spectra of the naphthaleneboronates included a pseudomolecular ion [M -i- H]" and a fragment ion [M -i- H-H20]. The most abundant ion from the fragmentation of BL derivatives is the parent ion, while the [M -I- H-H20] ion is the most abundant in the mass spectra of CS, teasterone and typhasterol. Full-scan mass spectra were readily obtained from 400 ng of free BRs, whereas the limit of detection in SIM mode was around 2 ng. The most... 

The HPLC method most widely used has been a reversed phase chromatography, with diode array detection enabling the detection of all microcystins bases in their UV spectra (Lawton 1994). This method offers a good separation of microcystins under the gradient elution conditions nevertheless, the lack of standards and certified reference materials makes necessary that the quantitation of these toxins had to be carried out based on purified MC-LR to give MC-LR equivalence. 

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