HPLC retention

In Section 2.1 the main chromatographic descriptors generally used in routine HPLC work were briefly discussed. Retention factor and selectivity are the parameters related to the analyte interaction with the stationary phase and reflect the thermodynamic properties of chromatographic system. Retention factor is calculated using expression (2-1) from the analyte retention time or retention volume and the total volume of the Uquid in the column. Retention 

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Avilamycins. At least 16 avilamycins have been reported (14,15,29) and are Hsted in Table 2. Avilamycins A (mp 181—182) and C (mp 188—189) are the primary components (14) produced by the strain Streptomjces viridochromogenes. Stmctures have been estabhshed based on chemical degradation, nmr, and x-ray analysis. Stmctures for the rest of the compounds have been assigned based on nmr and fab ms (14) interpretations. Avilamycins F and H have only one chlorine atom in the phenoHc ring. Relative hplc retention times for all the avilamycins have been recorded (14). 

The solvent used was hexane-isopropanol(4 1). Later, Allenmark and colleagues133 obtained enantioselective HPLC retention of a series of alkyl carboxymethyl sulphoxides (and other sulphoxides and classes) on a column of 3,5-... 

Note - Unequivocal identification of a total unknown using a single HPLC retention characteristic (or indeed a single retention characteristic from any form of chromatography) should not be attempted. 

In addition, synthesis of reference compounds and a comparison of HPLC retention characteristics and mass spectral data is often reqnired. 

Yasumoto et al. (30) describe two components of a Pseudomonas sp. culture with identical HPLC retention times to TTX and anhydro-TTX. These fractions produced typical signs of TTX intoxication in mice, with median death times similar to standard TTX and anhydro-TTX. Noguchi et al. (32) demonstrate by HPLC and GC-MS analyses that 7 biotypes of Vibrio sp. produced substances with retention times and molecular weights similar to TTX and anhydro-TTX. However, they observed mouse toxicity in only 1 biotype. Likewise, Simidu et al. (34) report that extracts of V. alginolyticus ATCC 17749 cultures displayed TTX-like toxicity in mice. The latter study shows that a variety of marine bacteria, plus E. coliy produced substances that, by HPLC analysis, were identical to TTX and anhydro-TTX. 

The solvent used was hexane-isopropanol(4 1). Later, Allenmark and colleagues obtained enantioselective HPLC retention of a series of alkyl carboxymethyl sulphoxides (and other sulphoxides and classes) on a column of (R)-lV-(3,5-dinitrobenzoyl)phenylglycine covalently bound via an amide bond (CSP 1), or ionically bound (CSP 2), to 3-aminopropylsilica the mobile phase was again hexane-isopropanol(4 l, or, also 19 1) and determination carried out at 254 or 280nm. The sulphoxides were better resolved on CSP 2. 

Buszewski, B., Gadza-la-Kopciuch, R. M., Markuszewski, M. L, Kaliszan, R. Chemically bonded silica stationary phases synthesis, physicochemical characterization, and molecular mechanism of reversed-phase HPLC retention. Anal. Chem. 1997, 69, 3277-3284. 

Valko, K., Du, C. M., Bevan, C., Reynolds, D. P., Abraham, M. H. Rapid method for the estimation of octanol/water partition coefficient (logP ,) from gradient RP-HPLC retention and a hydrogen bond addity term (EaJ ). Curr. Med. Chem. 2001, 8, 1137-1146. 

J.R.M. Smits, W.J. Meissen, G.J. Daalmans and G. Kateman, Using molecular representations in combination with neural networks. A case study prediction of the HPLC retention index. Computers Chem., 18 (1994) 157-172. 

Table 2 SRM scan table and HPLC retention times...

Reagents were purchased from commercial sources and used without further purification. Product identities were confirmed by comparison of HPLC retention times with authentic samples. The Pd/C catalyst was purchased as a dry, edge-coated, unreduced catalyst with 5 wt% in Pd. HPLC analyses were performed on a HP 1090 (stationary phase C18, 25 cm x 0.46 cm mobile phase CH3CN/H20 1% H3P04, 90% H20 to 100% CH3CN gradient over 25 min, 1 ml/min flow, UV-Vis detection). For safety aspects of handling Pd/C, see ref. 7.7... 

Interpretation of measurements of methods X-ray fluorescence spectrometry (Janssen and van Espen [1986] Arnold et al. [1994]), X-ray diffraction spectra (Adler et al. [1993]), NMR spectra (HIPS, Wehrens et al. [1993a]), HPLC retention indices (RIPS, Wehrens [1994]), Karl Fischer titration (HELGA, Wunsch and Gansen [1989]). 

These incubations are often carried out at 37 °C for 1-2 h. At different time points, 20-200 /aL of incubation mixture is withdrawn from each incubation and mixed with equal volume of ice-cold acetonitrile by vortexing. For preparation of acyl glucuronide, ice-cold acetonitrile containing 1% of formic acid is used to minimize acyl-migration [3,14]. After centrifugation at 13 000 rpm for 5-15 min, the supernatant (10-30 /aU) is analyzed by high-performance liquid chromatography (HPUC)-UV-MS. The metabolite of interest is identified based on HPLC retention time, UV spectrum and MS/MS data. Conversion yield is estimated based on UV absorption peak areas. A suitable in vitro enzyme system for scale-up is then... 

When using microbial products for mammalian metabolite identification, it is suggested to compare all the analytical data available. For example, slight differences in MS2 or MS3 spectra may indicate that the microbial products are not the same as the mammalian metabolite. Owing to matrix effects, HPLC retention time often varies from run to run, so it is good practice to spike a comparable amount of purified microbial product into the in vitro, in vivo or purified samples that contain the mammalian metabolite of interest. If the microbial metabolite and the mammalian metabolite are the same compound, then they should co-elute under different HPLC conditions, including different solvent pH, and the MS and/or UV peak area would increase accordingly. 

The quantitation of products that form in low yields requires special care with HPLC analyses. In cases where the product yield is <1%, it is generally not feasible to obtain sufficient material for a detailed physical characterization of the product. Therefore, the product identification is restricted to a comparison of the UV-vis spectrum and HPLC retention time with those for an authentic standard. However, if a minor reaction product forms with a UV spectrum and HPLC chromatographic properties similar to those for the putative substitution or elimination reaction, this may lead to errors in structural assignments. Our practice is to treat rate constant ratios determined from very low product yields as limits, until additional evidence can be obtained that our experimental value for this ratio provides a chemically reasonable description of the partitioning of the carbocation intermediate. For example, verification of the structure of an alkene that is proposed to form in low yields by deprotonation of the carbocation by solvent can be obtained from a detailed analysis of the increase in the yield of this product due to general base catalysis of carbocation deprotonation.14,16... 

High-pressure liquid chromatography (HPLC) - retention data (Locke 1974, Whitehouse and Cooke 1982,... 

HPLC retention time data have been used as a pseudo-molecular descriptor by Whitehouse and Cooke (1982), Hafkenscheid and Tomlinson (1981), Tomlinson and Hafkenscheid (1986) and Swann et al. (1983). 

Combination of HPLC retention data and molecular connectivity indices (Finizio et al. 1994). 

Calculation with HPLC retention data (Swann et al. 1983) ... 

The emphasis in this handbook is on experimentally determined values rather than estimated values. The latter are included when there is a lack of experimental data. Included in the experimental data are indirect measurements using GC or HPLC retention times. 

Vowles, P.D., Mantoura, R.F.C. (1987) Sediment-water partition coefficients and HPLC retention factors of aromatic hydrocarbons. Chemosphere 16, 109-116. 

Figure 3.5 Three-dimensional display of the photodiode array absorbance data obtained by HPLC/PDA/MS for a M. truncatula extract. The first dimension is HPLC retention time, second is wavelength, and third is absorbance. The data can be rapidly previewed for specific absorbance regions characteristic of functional groups.

HPLC/PDA/MS analysis of an alfalfa root extract. The HPLC retention time, the UV absorbance spectrum, and the mass spectrum readily identify the peak eluting at 45 minutes as medicarpin, a known phytoalexin in alfalfa. 

There have been several reports where plasma protein binding data was used in the prediction of in vivo properties of compounds. Two papers noted that the ability to predict in vivo clearance from in vitro microsome data was greatly improved when a plasma protein binding term was included [64,65]. In another study, binding to phospholipids and human serum albumin was assessed by HPLC retention times (on IAM and HAS columns, respectively) and used to predict volume of distribution [66]. 

HPLC retention times Partition coefficients can also be derived from retention times in high-pressure liquid chromatography (HPLC) analyses This approach provides some experimental advantages that simplify the analytical procedures and allow the handling of mixtures... 

The HPLC system used a mobile phase consisting of CH3CN/H2O/HCOOH (20 80 1, v/ v/v). Quantitation of standards and samples was achieved by isocratic elution over a C18,5 pm, Econosil HPLC column at a flow rate of 0.4 mL min. HPLC retention volumes and detection wavelengths for standards were as follows vanrllyl alcohol, 1.7 mL and 277 nm vanillic acid, 2.5 mL and 260 nm and vanillin, 4.1 mL and 284 nm. 

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