HPLC-ED Analysis

HPLC-ED analysis of nicotine (Figure 6.61) in dog plasma (0.1 mL) after protein precipitation with acetonitrile has been described." An ODS-modified silica column was used with (acetonitrile-methanol, 3 + l)-aq. sodium dihydrogen orthophosphate (2 mmol L ) containing sodium octyl sulfate (0.25 mmol L ) (1 + 19) (final pH 3). Detection was at PGEs (Ei +0.45 V, E2 +0.75 V V5 Pd). A LoD of 20pgL was... 

HPLC Techniques with Electrochemical Detection 95 Table 4.3 HPLC-ED analysis of amino acids... 

HPLC Techniques with Electrochemical Detection 99 Table 4.5 HPLC-ED analysis of natural phenolics... 

HPLC Techniques with Electrochemical Detection 101 Table 4.7 HPLC-ED analysis ofmycotoxins... 

Agricultural and Food Electroanalysis Table 4.8 HPLC-ED analysis of antibiotics... 

R. God and H. Gumm, Parallel HPLC in high-throughput analysis and puriflca-tion, in B. Yan (ed.). Analysis and Purification Methods in Combinatorial Chemistry, John Wiley Sons, Hoboken, NJ, 2004, pp. 307-320. 

The majority of the procedures listed have been applied to HPLC-ED in research publications, although they are rarely employed in routine analysis. Therefore only those of relevance to HPLC-ED are discussed here. 

Thiols are easily oxidised using a mercury electrode, but the analysis of thiol drugs poses special problems and is discussed in Chapter 5. Solutions of pheno-thiazines, particularly those without an electron withdrawing substituent at the 2-position, become coloured on storage due to oxidation. EC oxidation of these compounds at carbon electrodes is possible at +0.8 V vs Ag/AgCl. Dihydro-pyridines and other reduced aromatic compounds are also often oxidizable at moderate potentials and HPLC-ED methods for nifedipine, other 1,4-dihydro-pyridines, and pirprofen, a pyrroline derivative which can be oxidised to a pyrrole at + 0.8 V vs Ag/AgCl, have been described. 

Thiols disappear from urine in contact with air, forming both homo- and mixed-disulfides. Rapid losses have been reported for cysteine, D-penicillamine, and thiomalate. It is therefore essential to acidify urine immediately after it has been voided. Collection of urine into 10 mL of 6 mol L hydrochloric acid per litre of urine inhibits thiol oxidation for at least 12 h at room temperature. Although addition of EDTA and storing at —20 °C inhibits thiol degradation, further loss is still possible. Dithiothreitol (DTT) can be used to maintain thiols in the reduced form prior to analysis, but this procedure can give erroneous results since DTT might release thiols from mixed disulfides. Furthermore, DTT often interferes with HPLC-ED methods. 

The aim of this section is to discuss applications of HPLC-ED in the analysis of drugs and their metabolites in biological specimens, usually plasma. Analytes (International Nonproprietary Names, INNs are used when possible) are listed mainly by pharmacological category. Chemical structures are given for most analytes and internal standards, as are ED conditions, column, eluent, sample size and limit of detection (LoD) and low limit of quantitation (LLoQ). Brief details of sample preparation procedures are also included in many instances. 

Imipramine, desipramine (Figure 6.18) and their 2-hydroxy metabolites have been measured in plasma after LLE into diethyl ether.8-Hydroxyclomi-pramine was the internal standard. The analysis was performed on an ODS-modified silica column with acetonitrile-aq. acetate buffer (0.1molL pH 4.2) (40 + 60) containing sodium heptanesulfonate (5mmolL ) as eluent and ED (GCE, + 1,05 V vs Ag/AgCl). The LoDs were approximately 5)igL for each compound (1 mL sample). Koyama et al have also reported the assay of imipramine and its metabolites (including imipramine V-oxide) by HPLC-ED... 

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