Journal of Chromatography B (v.774, #2)

News Section (N1-N2).

An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 °C for 60 min), plasma (600 μl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C18 cartridge. Matrix components are eliminated with 2×500 μl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. LPV and NVP are eluted with 3×500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 μl MeOH 50%. A 40-μl volume is injected onto a Nucleosil 100, 5 μm C18 AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 μg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1–6.6% and 0.9–1.7% for LPV and NVP, and accurate (range of inter-day deviations −1.1 to +2.4%, and −1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.
Keywords: Lopinavir; Nevirapine;

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 μg mL−1) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid–liquid extraction. A Kromasil ODS 3 μm (150×3.2 mm I.D.) column was maintained at 40 °C and used with a mobile phase consisting of methanol–water (55:45, v/v). Fluorimetric detection was at an λ ex of 365 nm and an λ em of 445 nm. The limits of quantitation for amoxicillin were 0.1 μg mL−1 for gastric juice aspirate (500 μL), 0.5 μg mL−1 for plasma (50 μL) and 0.075 μg g−1 for gastric tissue (250 mg). The method was linear up to at least 15 μg mL−1 in gastric juice aspirate, up to 200 μg mL−1 in plasma and up to 100 μg g−1 in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.
Keywords: Amoxicillin;

Using 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a new derivatization reagent for HPLC and TLC, novel methods are described to detect secondary amine-bearing antidepressants (paroxetine, desipramine, fluoxetine, nortriptyline, maprotiline). The HPLC method is sensitive enough to detect these drugs in plasma at therapeutic levels whereas the latter has potential to detect them in overdose or forensic cases. The methods are based on purple chromogens formed by the displacement reaction of the drugs with TCNQ. The resulting chromogens are directly separated by either reversed-phase HPLC on a C18 column or TLC on silicagel plates. For HPLC, acetonitrile–water (60:40) was used as mobile phase, with detection at 567 nm and separation in 40 min. For TLC, three developing solvent systems were used. By HPLC, 36 ng ml−1 spiked plasma concentration of the drugs gave easily detectable signals whereas by TLC, detection limits varied mostly between 240 and 480 ng ml−1. The HPLC method was applied to real plasma samples. The methods described are simple and very selective; some metabolites of these antidepressants and a vast number of drugs do not interfere with detection.
Keywords: 7,7,8,8-Tetracyanoquinodimethane; Paroxetine; Desipramine; Fluoxetine; Nortriptyline; Maprotiline;

High-performance liquid chromatographic determination of 2-hydroxypropyl-γ-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence by Julianna Szemán; Andrea Gerlóczy; Katalin Csabai; József Szejtli; Georg L Kis; Ping Su; Raymond Y Chau; Albert Jacober (157-164).
A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-γ-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5–15 μg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.
Keywords: 2-Hydroxypropyl-γ-cyclodextrin;

Liquid chromatographic determination of triethylenetetramine in human and rabbit sera based on intramolecular excimer-forming fluorescence derivatization by Yukitaka Nakano; Hitoshi Nohta; Hideyuki Yoshida; Tetsuya Saita; Hiroshi Fujito; Masato Mori; Masatoshi Yamaguchi (165-172).
A highly selective and simple fluorimetric liquid chromatographic method for the determination of triethylenetetramine (TETA), a therapeutic drug for Wilson’s disease, in human and rabbit sera is described. This method is based on intramolecular excimer-forming fluorescence derivatization, which allows spectrofluorometric discrimination of polyamino compounds from monoamino species, followed by liquid chromatography. TETA and 1,6-hexanediamine (internal standard) were converted to the corresponding excimer-forming derivatives with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester. The derivatives were separated within 20 min on a reversed-phase column using isocratic elution and detected spectofluorometrically at 480 nm with excitation at 345 nm. This method was successfully applied to the monitoring of TETA in human and rabbit sera with a simple pretreatment. The detection limit for TETA in serum was 18 ng/ml (0.13 nmol/ml) corresponding to 0.2 pmol on column at a signal-to-noise ratio of 3.
Keywords: Triethylenetetramine;

Validated method for quantitation of biomarkers for benzene and its alkylated analogues in urine by J.B Laurens; X.Y Mbianda; J.H Spies; J.B Ubbink; W.J.H Vermaak (173-185).
A validated gas chromatography–mass spectrometric method for the analysis of the metabolites of benzene and its alkylated analogues in urine is reported. A number of metabolites, as required by authorities for biomonitoring of industrial exposure to aromatic vapour, were analysed simultaneously with preservation of quantitative information concerning positional isomers. The use of this method replaces a combination of analytical methods required for the analysis of all these metabolites. Urine samples were subjected to acidic deconjugation followed by a derivatization step. Phenol, ortho-, meta-, para-cresol, mandelic acid, and ortho-, meta-, para-methylhippuric acid were analysed as their corresponding ethoxycarbonyl derivatives, with single ion monitoring. The mass-to-charge ratios (m/z) of the ions used for quantitation by single ion monitoring of the metabolites were: phenol, 94 m/z; cresols, 108 m/z; mandelic acid, 206 m/z; hippuric acid, 105 m/z; methylhippuric acids, 119 m/z. The mass-to-charge ratios for the internal standards were: [2H6]phenol, 99 m/z; p-chlorophenol, 128 m/z and 3-chloro-4-hydroxyphenyl acetic acid, 214 m/z. The limits of detection for phenol and the cresols were below 0.4 μmol/l and below 0.05 μmol/l for mandelic acid and the hippuric acids. Within-run precision for mandelic acid was 6.2%, for hippuric acid was 7.32% and was below 5% for the rest of the analytes.
Keywords: Benzene; Phenol; Cresol; Methylhippuric acid;

A novel, reversed-phase high-performance liquid chromatographic (HPLC) method is described for the analysis of sirolimus (SRL) in whole blood. The samples were purified by precipitating blood matrix with zinc sulfate, SRL was then extracted with acetone followed by solid-phase extraction. The method was linear over a range of 1–100 ng/ml and the lower limit of quantification was 2.5 ng/ml. The coefficient of variation (within day) was below 8.0% for the lowest SRL concentration. The day-to-day coefficient of variation was below 6.6%. The assay did not show interference peaks with immunosuppressive drugs commonly given to transplant patients. With the simplified extraction procedure described, 60 samples (including controls and calibration curve) can be quantified in a day. The sensitivity and rapidity of this analytical procedure makes it useful for routine therapeutic monitoring of SRL.
Keywords: Sirolimus;

A sensitive and selective method is described for the determination of artemether and its active dihydroartemisinin metabolite in human plasma using artemisinin as internal standard. The method consists of a liquid–liquid extraction with subsequent evaporation of the supernatant to dryness followed by the analysis of the reconstituted sample by liquid chromatography–mass spectrometry (LC–MS) in single ion monitoring mode using atmospheric pressure chemical ionization (APCI) as an interface. Chromatography was performed on a C18 reversed-phase column using acetonitrile–glacial acetic acid 0.1% (66:34) as a mobile phase. The method was fully validated over a concentration range of 5–200 ng/ml using 0.5 ml of human plasma per assay. Stability assessment was also included. The method was applied to the quantification of artemether and its metabolite in human plasma of healthy volunteers participating in pharmacokinetic drug–drug interaction studies.
Keywords: Artemether; Dihydroartemisin;

Simultaneous quantification of prostaglandins, isoprostane and thromboxane in cell-cultured medium using gas chromatography–mass spectrometry by Hiroki Tsukamoto; Takanori Hishinuma; Tsuyoshi Mikkaichi; Hironori Nakamura; Tohru Yamazaki; Yoshihisa Tomioka; Michinao Mizugaki (205-214).
We have developed a simultaneous quantification method for prostaglandin (PG) E2, PGD2, PGF, 8-epi-PGF, 6-keto-PGF and thromboxane (TX) B2. Using [3,3,4,4-2H4]PGE2, [3,3,4,4-2H4]PGD2, [3,3,4,4-2H4]8-epi-PGF, [3,3,4,4-2H4]PGF, [3,3,4,4-2H4]6-keto-PGF and [18,18,19,19-2H4]TXB2 as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography–mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.
Keywords: Prostaglandins; Isoprostane; Thromboxane;

When the assumption of homoscedasticity is not met for analytical data, a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line is to use weighted least squares linear regression (WLSLR). The purpose of the present paper is to stress the relevance of weighting schemes for linear regression analysis and to show how this approach can be useful in the bioanalytical field. The steps to be taken in the study of the linear calibration approach are described. The application of weighting schemes was shown by using a high-performance liquid chromatography method for the determination of lamotrigine in biological fluids as a practical example. By using the WLSLR, the accuracy of the analytical method was improved at the lower end of the calibration curve. Bioanalytical methods data analysis was improved by using the WLSLR procedure.

5-Fluorouracil (5-FU) is a commonly used anti-cancer drug with notable activity in clinical practice, yet it causes significant unpredictable and often serious toxicity. Both 5-FU and uracil (U) are catabolised by dihydropyrimidine dehydrogenase (DPD) to form dihydrofluorouracil (FUH2) and dihydrouracil (UH2), respectively. A means of predicting toxicity before treatment would be more valuable. Variations in dihydropyrimidine dehydrogenase (DPD) activity between patients are at least partly responsible for variable toxicity. Measurement of the UH2 to U ratio may be a measure of pyrimidine catabolism and thus be utilised to predict subsequent toxicity. We have developed an efficient extraction and detection method using HPLC for the simultaneous measurement of UH2 and U in plasma. A single C18 Spherisorb ODS2 (25 cm) column using isocratic elution was utilised. U, UH2 and the internal standard 4-chlorouracil were detected at wavelengths of 257, 220, and 268 nm, respectively. The chromatographic run time was 45 min which is half that of other methods. The detection limit was 0.02 μM for U and 0.1 μM for UH2 using only 0.5 ml of plasma for both compounds. The basal plasma concentrations of U and UH2 in 23 individuals ranged from 0.025 to 0.27 μM and 0.4–1.7 μM, respectively. This simple method may permit the assessment of pyrimidine catabolism, and therefore allow prediction of the toxicities associated with the use of fluorinated pyrimidines.
Keywords: Uracil; Dihydrouracil; 5-Fluorouracil;

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC–MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 μl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC–MS 9,9,9-D 3-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring-d 5, 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at −80 °C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at −80 °C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.
Keywords: 4-Hydroxynonenal;