Reversed-phase HPLC reference standards

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

The metabolites of interest in urine are separated using reverse-phase HPLC combined with electrospray ionization (ESI)-MS/MS, and detection is performed using multiple-reaction monitoring. Stable-isotope-labeled reference compounds are used as internal standards. 

The extraction protocol typically recovers 96% to 108% of isoflavones. The HPLC protocol typically recovers isoflavones in a range between 97% and 105% when tested using spiked reference standards. During reversed-phase HPLC separation, all isoflavones elute from the column in the order of polar to nonpolar and are separated within 50 min of run time. 

Pearce and Lushnikova [78] used semipreparative HPLC method for the isolation of the three omeprazole metabolites produced by the fungi. Incubation of Cunninghamella elegans ATCC 9245 and omeprazole allowed putative fungal metabolite to be isolated in sufficient quantities for structural elucidation. The metabolites structures were identified by a combination of LC/MS and NMR spectrometric experiments. These isolates are used as reference standards in the confirmatory analysis of mammalian metabolites of omeprazole. In the LC/MS and LC/MS/MS analysis, components were separated by reversed-phase HPLC on a Hypersyl HyPurity (15 cm x 4.6 mm, 5 /im) column. The mobile phase consisted... 

The decarboxylated product of moxalactam (see Section 4) is determined by an HPLC technique. A reverse phase HPLC system consisting of 80 parts of 0.1M ammonium acetate and 20 parts of methanol is used with a Dupont Zorbax C8 or other suitably similar column to determine the decarboxylated product. In this system, the decarboxylated moxalactam should elute with a k of about 6.5. The decarboxylated moxalactam is quantitatively determined by comparing the peak response for the sample with a peak response calibration curve of the authentic decarboxylated moxalactam reference standard material. 

A comprehensive semi-empirical description of reversed-phase HPLC systems, for predicting the relative retention and selectivity within a series of analytes, has been developed by Jandera and co-workers [72,73]. The approach consists of determining the interaction indices and the structural lipophilic and polar indices. A suitable set of standard reference analytes is necessaiy to calibrate the retention (or selectivity) scale. 

The predominant mode of HPLC, reversed phase, involves the separation of material based on the partitioning between a relatively polar mobile phase and a nonpolar stationary phase. Normal phase HPLC—nonpolar mobile phase and polar stationary phase—is considered an orthogonal technique to reversed-phase HPLC when qualifying reference standards. In fact it is common for the elution order to be entirely reversed when switching an analysis from reversed to normal phase. Therefore, highly nonpolar impurities can be easily characterized by normal phase separations. 

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. 

In order to establish that a process results in consistent product, analytical tests should be established and qualified by using them to test a reference standard. These tests are developed as in-process test methods to track the quantity and quality of product during optimization of the purification process. In-process analyses often involve using ion exchange HPLC, reverse phase HPLC, SDS-PAGE, Western blot, and/or ELISA assays. 

Gerrits et al. (20) determined thiamine and its phosphates in whole blood by reversed-phase HPLC with precolumn derivatization. Good resolution of the elution profile for thiamine and its phosphate esters was obtained for a standard sample (Fig. 8) and for a human blood sample (Fig. 11). Reference values obtained from the whole blood samples of 65 healthy volunteers were in nM (mean SD) thiamine, 4.3 1.9 TMP, 4.1 1.6 TPP, 120 17.5. For TTP, all measurements were <4.0 nM. However, a few patients with low TPP and relatively high TTP were found, and in most cases these patients were alcoholics. The precise role of TTP in a clinical setting remains to be elucidated. 

In order to study simultaneously the behaviour of parent priority surfactants and their degradation products, it is essential to have accurate and sensitive analytical methods that enable the determination of the low concentrations generally occurring in the aquatic environment. As a result of an exhaustive review of the analytical methods used for the quantification within the framework of the three-year research project Priority surfactants and their toxic metabolites in wastewater effluents An integrated study (PRISTINE), it is concluded that the most appropriate procedure for this purpose is high-performance (HP) LC in reversed phase (RP), associated with preliminary techniques of concentration and purification by solid phase extraction (SPE). However, the complex mixtures of metabolites and a lack of reference standards currently limit the applicability of HPLC with UV- or fluorescence (FL-) detection methods. 

HPLC Assay of an ethyl alcohol-acetic acid extract against a Reference Standard material (chromatographic conditions reversed-phase mode Cjo column methanol—water—acetic acid, 50 49 1 (v/v), mobile phase detection at 254 nm [79]) and (c) extraction of tablets with methanol or ethanol and UV measurements at 260 or 262 nm. Linearity in the 3—15 Ug/ml and standard errors of less than 2% were reported [65,109,110]. A similar procedure is used for the determination of the Content Uniformity of the tablets [79].. ... 

Proteolytic secretion variants of recombinant human growth hormone were isolated by anion-exchange HPLC but compared analytically to the reference standard using reversed-phase (C4) HPLC with an ammonium bicarbonate/acetonitrile mobile phase rather than the more commonly used TFA/acetonitrile mobile phase [297]. 

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. 

Alkyl-Type Phases (C1-C18, C30). Probably 90% of all reversed-phase columns are alkyl-type bonded phases. An enormous amount of publications are devoted to the classiflcation, standardization, and comparison of these phases. In their book Practical HPLC Method Development, Snyder and Kirkland [57] indicate that reversed-phase retention for nonpolar and nonionic compounds generally follows the retention pattern Cl < C4 < C8 = Cl 8. At the same time, they refer to the comparison of Cl 8-type columns from different manufacturers and find dramatic variation in the retention of both polar and nonpolar compounds at the same conditions on different columns. 

RP-TLC and HPLC provide a variety of descriptors that can be used as lipophilicity indices. Among them, extrapolated capacity factors often lead to 1 1 correlation with octanol-water log P. On the other hand, isocratic capacity factors need fewer experiments to be determined however, they depend strongly on chromatographic conditions. The CHI combines easy and rapid measurements with a uniform lipophilicity scale. However, both h-pophilicity and reversed-phase chromatographic retention are composite phenomena and, consequently, their resemblance cannot always be anticipated. Because standard reference sets cannot be available for all strucmrally diverse compounds, a comparison between chromatographic indices and octanol-water log P within the series of the investigated compounds is still indispensable. 

Another novel approach that could be used to quantify metabolites in the absence of reference standards or radiolabel was described by Ramanathan et al. [78], In this approach, recently introduced NSI technique and two HPLC systems were coupled with a Q-TOF mass spectrometer to obtain normalized LC-MS response of drugs and their metabolites. As shown in Figure 5.12, one HPLC system performs the separation of a drug and its metabolites, while the other system adds solvent postcolumn with an exact reverse of the mobile phase composition so that the final composition entering the NSI source is isocratic throughout the entire HPLC run. 

Fig. 10.3 Reversed-phase high-performance liquid chromatography (HPLC) of Me3C(CH=CH) jCMe3 prepared by the polymerization of acetylene (5 equivalents) in toluene, initiated by W(=CHCMe3)(=NAr)(OCMe3)2 (1 equivalent) in the presence of quinuclidine (5 equivalents) and terminated by Me3CCHO. The peak marked 3t3 is for the oligomer with jc = 3 and 3 trans double bonds likewise for the other marked peaks. The unmarked peaks refer to oligomers containing at least one cis double bond. The peak marked with an asterisk is the internal standard (guaiazulene) (Schlund 1989).

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