Journal of Chromatography B (v.795, #2)

OFC: Update (OFC).

News Section (N1-N2).

A sensitive, rapid, selective and reproducible method has been developed to measure blood plasma levels of benzidine (BZ) and its acetylated metabolite, N-OH-N,N′-diacetylbenzidine (N-OH-DABZ), using supercritical fluid chromatography (SFC) for the first time. Benzidine and N-OH-N,N′-diacetylbenzidine were extracted from the plasma using ether. Separation was done on a Nucleosil (250  mm×4.6  mm) 10 μm, Nucleosil-RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml min−1) as mobile phase. The column temperature was 45 °C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 280 nm. The limit of quantification was 0.10 ng ml−1 (BZ) and 0.14 ng ml−1 (N-OH-diacetylbenzidine) using 1 ml plasma specimen. The mean extraction recovery of BZ was found to be 98.6%. The SFC method was directly compared to a published HPLC–UV method. With respect to speed, organic solvent usage, sensitivity, specificity and accuracy, SFC was found to be superior. The method has been successfully used to estimate the BZ, N-OH-diacetylbenzidine levels in blood plasma of the animals who were administered 15 μg kg−1 body weight of benzidine.Further, this method has been also applied for the detection and quantification of benzidine DNA and hemoglobin adducts from the blood and tissue samples of the benzidine dosed animals.
Keywords: Benzidine; Diacetylbenzidine;

Stability studies of selected doping agents in urine: caffeine by R Ventura; C Jiménez; N Closas; J Segura; R De la Torre (167-177).
The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid–liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4±6.0 and 87.3±5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6–30 μg/ml (r 2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 μg/ml, respectively. Precisions in the range of 1.5–9.2 and 4.1–5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 μg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 °C and in liquid urine samples stored at 4, −20, −40 and −80 °C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 °C for 7 days. The effect of repeated freezing (at −20 °C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at −20 °C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.
Keywords: Caffeine;

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine by Joo Youn Cho; Hyeong Seok Lim; Kyung Sang Yu; Hyun Joo Shim; In Jin Jang; Sang Goo Shin (179-186).
A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid–liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 μl of 0.1 M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150  mm×4.6  mm I.D., 5 μm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20 mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were ≤15 and ≤10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.
Keywords: Phosphodiesterase inhibitor; DA-8159;

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography–tandem mass spectrometry (LC–MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1–2 compounds using the conventional methods.
Keywords: Plasma protein binding; High-throughput; Proteins;

Human biomonitoring of pyrethrum and pyrethroid insecticides used indoors: by L Elflein; E Berger-Preiss; A Preiss; M Elend; K Levsen; G Wünsch (195-207).
This work describes a gas chromatographic-mass spectrometric method employing negative chemical ionization (NCI) for the determination of E-cis/trans-chrysanthemumdicarboxylic acid (CDCA) in human urine used as a biomarker for the exposure to pyrethrum and/or certain pyrethroids in insecticide formulations applied indoors. Mixed-mode solid phase extraction was utilized for sample cleanup. Extraction recoveries ranged from 92 to 104% (2–9% R.S.D.). The acids were esterified with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) allowing both their gas chromatographic separation and their sensitive mass spectrometric detection under NCI conditions. Detection limits of ca. 0.05 μg/l urine were achieved.
Keywords: Derivatization, GC; Pyrethrum; Pyrethroid insecticides; Chrysanthemumdicarboxylic acid;

A simple and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of cyclosporine (CyA, also known as cyclosporin A) in human whole blood. The method entailed direct injection of the blood samples after deproteination using acetonitrile. Chromatography was carried out using an ODS column under isocratic elution with acetonitrile–5 mM disodium hydrogen phosphate (75:25, v/v), pH 5.1 at 70 °C and a detector set at 210 nm. The mean absolute recovery of cyclosporine from blood was 97%, and the linearity was assessed in the range of 100–3000 ng/ml blood, with a correlation coefficient of greater than 0.999. The limit of quantification and detection of the present method were 100 and 50 ng/ml, respectively. This method has been used to analyze several hundred human blood samples for bioavailability studies.
Keywords: Cyclosporine;

A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 μl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis® HLB column (1  mm×50  mm, 30 μm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column (4.6  mm×50  mm, 5 μm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/x 2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at −30 °C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos® formulation (encapsulated 15 mg tablet) and an Actos® 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos® 15 mg tablet.
Keywords: Bioequivalence study; Pioglitazone;

Simple and efficient method for enantioselective determination of omeprazole in human plasma by Ricardo Mathias Orlando; Pierina Sueli Bonato (227-235).
A practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma is presented. C18 solid phase extraction (SPE) cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol:hexane (70:30) as the mobile phase, at a flow rate of 0.5 ml/min. The detection was carried out at 302 nm. The method proved to be linear in the range of 10–1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%.
Keywords: Enantiomer separation; Omeprazole;

Determination of anti-cancer drug actinomycin D in human plasma by liquid chromatography–mass spectrometry by Gareth J Veal; Julie Errington; Julieann Sludden; Melanie J Griffin; Lisa Price; Annie Parry; Juliet Hale; Andrew D.J Pearson; Alan V Boddy (237-243).
Actinomycin D is an anti-cancer drug commonly used in the treatment of paediatric malignancies such as Wilms’ tumour, Ewing’s sarcoma and rhabdomyosarcoma. Despite its long history of clinical use, little is known about the pharmacokinetics of actinomycin D in humans, largely due to problems in developing an analytical assay with the required sensitivity to measure relevant clinical concentrations. As actinomycin D treatment in children with cancer is associated with veno-occlusive disease (VOD), and as the dose intensity of actinomycin D treatment has been defined as a significant risk factor for the development of this potentially life-threatening hepatic toxicity, pharmacokinetic studies of actinomycin D may be beneficial in optimizing treatment with this drug. In order to investigate this issue, we developed a sensitive liquid chromatography–mass spectrometry (LC–MS) method for the determination of actinomycin D in human plasma samples. Extraction of analytical samples was carried out with acetonitrile and analysis performed on an API 2000 LC/MS/MS using an internal standard of 7-aminoactinomycin D. A limit of quantitation of 1.0 ng/ml was determined, allowing the reliable measurement of actinomycin D in plasma samples obtained from patients receiving this drug clinically. The method demonstrated good reproducibility, over the calibration curve range of 1.0–100 ng/ml, with intra- and inter-assay precision CVs of 2.7–11.3 and 2.3–7.8%, respectively. Accuracy data showed relative errors of 2.0–16.4 and 10.4–15.2% for intra-assay (n=10) and inter-assay (n=7) experiments, respectively. Initial results of actinomycin D pharmacokinetics in paediatric patients are shown.
Keywords: Actinomycin D;

A rapid on-line solid phase extraction/liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) method using restricted access material (RAM) was developed for the simultaneous determination of eight organophosphorus triesters in untreated human blood plasma. In a process involving column-switching techniques, the analytes were enriched on the RAM column, separated using a C-18 analytical column and detected with LC/MS. Tandem mass spectrometry was used to characterize and quantify the analytes. To elucidate the fragmentation pathway of a number of the analytes, MS3 experiments using an ion trap mass spectrometer were performed. The matrix effects associated with using APCI and ESI interfaces were investigated. The recoveries obtained were in the range 60–92% (R.S.D.<6%), with estimated detection limits between 0.2 and 1.8 ng/ml of plasma, and the total analysis time was 27 min.
Keywords: Restricted access material; Organophosphorus triesters;

A specific reversed phase-high pressure liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination of clozapine (CZP), loxapine (LXP), zuclopenthixol (ZPT) and flupenthixol (FPT) in plasma. These four antipsychotic drugs are frequently used for the treatment of schizophrenia and other neuropsychiatric diseases. Carpipramine, a dihydrodibenzazepine, was used as an internal standard (I.S.). A liquid–liquid procedure was used to extract the drugs from human plasma. The analysis was performed on a XTerra™ MS C18 column with UV detection. Calibration curves were linear in the range 50–1000 μg/l. The limit of quantification (LOQ) was 15 μg/l for clozapine and loxapine and 20 μg/l for zuclopenthixol and flupenthixol. The coefficient of variation (CV) for intra- and inter-day precision was 7.2% or less with accuracies within 10% for the three concentrations.This isocratic and rapid method (run time<10 min) is useful for the management of acute intoxication.
Keywords: Clozapine; Loxapine; Zuclopenthixol; Flupenthixol;

Liquid chromatographic determination of pyronaridine in human plasma and oral dosage form by Chinedum P Babalola; Gerhard K.E Scriba; Akin Sowunmi; Olaniyi A Alawode (265-272).
A new procedure for the determination of pyronaridine in plasma by reversed-phase high performance liquid chromatography (HPLC) with UV detection at 278 nm is described. The method involves liquid–liquid extraction of the drug with diethyl ether following basification of the deproteinized plasma with alkaline phosphate buffer. Chromatographic separation was achieved using a microbore C-18 column and a mobile phase consisting of 0.1% aqueous trifluoroacetic acid (TFA)–acetonitrile (75:25% (v/v)), pH 2.2, at a flow rate of 0.07 ml/min. Papaverine was used as internal standard. The response was linear between 50 and 1500 ng/ml. The limit of quantitation (LOQ) after plasma extraction was 50 ng/ml, the intra- and inter-day precision ranged from 2.5 to 13.8% (CV). The recovery of the drug from plasma and accuracy were >90%. Preliminary application of the method for monitoring pyronaridine in humans upon oral administration of the tablet demonstrated the principal usefulness of the assay for clinical trial studies. The method can also be used to analyze the compound in pharmaceutical formulations.
Keywords: Pyronaridine;

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore® C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/106 cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/106 cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94–104%. Atazanavir was stable in PBMC for at least 24 h at room temperature and for at least 129 days at −15 °C.
Keywords: Peripheral blood mononuclear cells; Atazanavir;

Oxaliplatin ([(1R,2R)-1,2-cyclohexanediamine-N,N′]oxalato(2-)-O,O′-platinum) is the first platinum drug with significant activity for metastatic colon cancer. The analysis of oxaliplatin has previously almost exclusively been based on the determination of the platinum content in plasma or ultrafiltrate using flameless atomic absorption spectroscopy (FAAS) or inductively coupled plasma mass spectrometry (ICPMS). A new method for quantitative determination of the free fraction of the intact drug in blood ultrafiltrate is presented here. Blood was ultrafiltrated centripetally at 4 °C and the ultrafiltrate was analyzed by liquid chromatography. Oxaliplatin was separated on a Hypercarb column using a mobile phase of methanol/succinic acid buffer pH 7.0 (9/1, v/v). Post-column derivatization was performed by adding N,N-diethyldithiocarbamate in methanol and with microwave heating of a Teflon tubing. The derivative was quantified by photometric detection at 344 nm. The coefficient of variation of standard blood samples was 4.9 and 2.5% at 0.100 and 1.00 μg/ml, respectively. The limit of quantitation was 0.04 μg/ml.
Keywords: Derivatization; Oxaliplatin;

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis® HLB extraction column (2.1  mm×20  mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00–1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1–94.7 and 90.1–96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20 mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.
Keywords: Methoxyamine; 4-(N,N-diethylamino)benzaldehyde;

A novel insoluble topoisomerase I inhibitor, 9-nitrocamptothecin (9-NC), is in advanced stages of clinical development and has been used to treat a diverse array of tumor types, including breast, ovarian, pancreatic and haematological malignancies. We have established a sensitive high-performance liquid chromatography method using fluorescence detection for the quantitation of 9-NC. Non-fluorescent 9-NC is converted to fluorescent 9-aminocamptothecin (9-AC) via a one-step pre-column derivative reaction. The quantitative limit of 9-NC was 1 ng/ml and the method was reproducible with the respective intra- and inter-day variability falling below 5.0 and 9.0%. The determination of both 9-NC and its metabolite 9-AC in dog plasma was also achieved using the same chromatographic and detection conditions. In dog plasma, the quantitative limits of 9-AC and 9-NC were 0.25 and 1 ng/ml, respectively. The presence of 9-AC in the samples yielded no interference with the determination of 9-NC. However, individual matrices can affect the conversion efficiency of 9-NC, thus indicating that standard samples should be run for each matrix.
Keywords: Derivatization, LC; 9-Nitrocamptothecin; 9-Aminocamptothecin;

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural compound with antioxidant properties of a new family of sulfur-containing amino acids. It has been detected in human urine and plasma, in mammalian cerebellum and, more recently, in dietary vegetables. In the present study, a simple, highly sensitive method using a high-performance liquid chromatography system with electrochemical detection (ECD) has been developed. The method showed excellent precision and accuracy. It has been found to be about 100-fold more sensitive than gas chromatographic method and 2000-fold more sensitive in respect to the liquid chromatography method with UV detection. The method showed the required features of specificity and sensitivity to detect aminoethylcysteine ketimine decarboxylated dimer in human plasma and in cultured cells after in vitro supplementation.
Keywords: Aminoethylcysteine ketimine decarboxylated dimer;

A simple micellar electrokinetic chromatography is described for well resolution of theophylline, dyphylline and caffeine. The separation was performed at 25 °C using a background electrolyte consisting of 10 mM borate buffer at pH 9 and 40 mM sodium dodecyl sulfate (SDS) as running buffer. Under this condition, good separation with high efficiency and short analyses time required is achieved. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the borate buffer and sodium dodecyl sulfate. Using caffeine as an internal standard (I.S.), the linear range of the method for the determination of theophylline and dyphylline was over 0.03–1 μmol ml−1; the detection limit (signal-to-noise ratio 3; injection 0.3 psi, 3 s) was 0.01 and 0.02 μmol ml−1, respectively.
Keywords: Theophylline; Dyphylline;

This paper describes the ultra-fast determination of a pharmaceutical compound using TurboIonSpray™ LC-MS-MS on an API 4000 mass spectrometer. Sample preparation consisted of plasma protein precipitation, centrifugation and dilution of the supernatant. The use of small analytical column dimensions (2.1  mm×10  mm) and high eluent flow rates (up to 2.2 ml/min) in isocratic mode led to a retention time of 9 s. A sample-to-sample cycle time of only 10 s was achieved by coupling two autosamplers. Partial separation of the drug and its main metabolite could be obtained. The d5-labeled drug used as internal standard compensated for matrix suppression effects. The assay was linear in the concentration range 1–1000 ng/ml, using standards prepared in human plasma. Inter-assay accuracy and precision were 98.5 and 6.2%, respectively. Mean intra-assay accuracy and precision calculated from quality control (QC) samples in human, rat and dog plasma at 3, 30 and 800 ng/ml were 100.8 and 3.8%, respectively. The ultra-fast LC-MS-MS method was successfully cross-validated against a commonly used column-switching LC-MS-MS assay with 2.3 min run time by analyzing real study samples.
Keywords: Ultra-fast quantitative bioanalysis;

Determination of urinary S-phenylmercapturic acid by liquid chromatography-tandem mass spectrometry by M. Pieri; N. Miraglia; A. Acampora; G. Genovese; L. Soleo; N. Sannolo (347-354).
Urinary S-phenylmercapturic acid (S-PMA) is considered a useful biomarker for the measurement of low levels of benzene exposure, related to occupational exposure, smoking habits or environmental pollution. S-PMA quantitative analysis requires highly sensitive and specific techniques and purification procedures, mainly based on liquid–liquid or solid-phase extraction, which result in time expensive analyses. A method was developed for the quantitative determination of S-PMA in urine by using a simple, reproducible and easily automatizable HPLC purification followed by LC/ESI-NI/MS2 analysis. In order to reduce the cost of the analysis, related to the use of expensive labeled standards, p-bromo-S-phenylmercapturic acid (p-Br-S-PMA) was synthesized, characterized and used as internal standard.The feasibility and efficacy of the proposed method were examined by constructing calibration curves in the range from 6.2 to 200 μg/l and data were analyzed in terms of linearity and statistical parameters. The detection limit, related to the purification of 1 ml urine sample is 5 μg/l. The method was applied to the analysis of 12 urine samples from smoker subjects non-occupationally exposed to benzene. S-PMA urinary levels ranged from 13.6 to >200 μg/l, suggesting a high influence of life style in the S-PMA excretion. The proposed analytical method is suitable for the biological monitoring of both smoker and non-smoker workers, occupationally exposed to benzene. By processing at least 2 ml of urine samples, the method appears to be also useful for the evaluation of benzene uptake due to the environmental pollution.
Keywords: Benzene exposure; Biological monitoring; S-Phenylmercapturic acid;

For the determination of pentacyclic triterpenes of the boswellic acid family in human plasma a novel sensitive method was developed combining serial extraction on diatomaceous earth and graphitized carbon black followed by reversed phase high-performance liquid chromatography (HPLC) and photodiode array detection. The overall average extraction yield of 12 different pentacyclic triterpenic acids was approximately 66%. The calibration graphs were linear with coefficients of correlation for all compounds greater than 0.999. The overall within-day and between-day coefficients of variation (CV) for the 12 pentacyclic triterpenic acids were 5.6 and 6.8%, respectively. This HPLC procedure delivers the analytical sensitivity, precision and accuracy required for clinical pharmacokinetic and therapeutic studies.
Keywords: Pentacyclic triterpenic acids;

Automated method for determination of glutardialdehyde residues in flexible endoscopes after disinfection by Monika Emmrich; Hans Floss; Birgit Zühlsdorf; Heike Martiny (363-370).
Glutardialdehyde (GDA) is the most commonly used disinfectant for flexible endoscopes. After inappropriate rinsing of endoscopes residual GDA in the narrow endoscope channels may lead to toxic effects in patients. Common methods for determination of aldehydes in water involve derivatization with 2,4-dinitrophenylhydrazine (DNPH), liquid–liquid or solid-phase extraction and HPLC determination. Since derivatization and extraction is both time-consuming and labor-intensive only a small number of samples can be measured. Thus, we developed a fully automated method which includes a conventional HPLC system, a programmable autosampler, and UV detection. After GDA derivatization using DNPH the samples remain in the aqueous phase and no preconcentration of the analyte is necessary. The samples are automatically derivatized through the autosampler. While derivatization in one sample takes place the previous sample is injected and measured by HPLC. Our method is well suited for screening residual GDA in endoscopes as it is both time- and labor-saving.
Keywords: Endoscopes; Glutardialdehyde;

Simultaneous determination of 3′-azido-2′,3′-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography by Linghui Kong; John S Cooperwood; Chang H Oh; Chung K Chu; F.Douglas Boudinot (371-376).
3′-Azido-2′,3′-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N 3-pivaloyloxymethyl-3′-azido-2′,3′-dideoxyuridine (I), 5′-pivaloyloxymethyl-3′-azido-2′,3′-dideoxyuridine (II), 5′-O-valinyl-3′-azido-2′,3′-dideoxyuridine hydrochloride (III) and 5′-O-phenylalanyl-3′-azido-2′,3′-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 μM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3′-Azido-2′,3′-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 μg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25–100 μg/ml for AZDU and prodrugs II, III, and IV and 0.5–100 μg/ml for prodrug I. The limit of quantitation was 0.25 μg/ml for prodrugs II, III and IV and 0.5 μg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.
Keywords: 3′-Azido-2′,3′-dideoxyuridine;

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of 5-fluorouracil (5-FU) and its prodrug, tegafur (TF), in dog plasma. 5-FU, the internal standard, 5-bromouracil, and TF were separated on a C18 Spherisorb ODS2 column using isocratic elution with retention times of 4.4, 8.0 and 21.2 min, respectively. Detection by UV absorption at 260 nm gave a limit of quantitation of 4 μg/l for 5-FU in plasma. Calibration curves for 5-FU and TF were linear over the ranges of 4–160 μg/l and 0.48–19.2 mg/l, respectively. Intra- and inter-day precision over these concentration ranges were <10.9 and <13.6% for 5-FU and TF, respectively, with good accuracy for both compounds. The method was successfully applied to define plasma concentration–time curves of TF and 5-FU in dogs administered a single oral dose containing TF (100 mg) and uracil (224 mg).
Keywords: 5-Fluorouracil; Tegafur;

Determination of irinotecan (CPT-11) and SN-38 in human whole blood and red blood cells by liquid chromatography with fluorescence detection by Floris A. de Jong; Ron H.J. Mathijssen; Peter de Bruijn; Walter J. Loos; Jaap Verweij; Alex Sparreboom (383-388).
An analytical method was developed for the anticancer agent irinotecan (CPT-11) and its main metabolite SN-38 in human whole blood and in red blood cells (RBCs). Sample pretreatment involved deproteinization of whole blood or plasma-diluted RBCs isolated by MESED instruments, with a mixture of aqueous perchloric acid and methanol (1:1, v/v). Separation was carried out using isocratic elution on a Hypersil ODS stationary phase, with detection at excitation and emission wavelengths of 355 and 515 nm, respectively. The lower limit of quantitation (LLQ) in blood was established at 5.00 ng/ml for both compounds, with values for within-run precision (WRP) and between-run precision (BRP) of less than 10%. The method is currently being applied to investigate the blood distribution of CPT-11 and SN-38 in cancer patients.
Keywords: Irinotecan; SN-38; CPT-11;

A method for the simultaneous determination of 2-methoxyphenol, 2-methoxy-4-methylphenol, 2,6-dimethoxyphenol and 4′-hydroxy-3′-methoxyacetophenone in urine has been described. The metabolites were analyzed after enzymatic hydrolysis and extraction on octyl (C8) cartridges by using gas chromatography with flame ionization detection and a 5/95% copolymer of diphenyl–poly(dimethylsiloxane) capillary column. Methoxyphenols were well separated within 12 min. Recovery was over 90% in the range from 0.5 to 20 μg/ml; the detection limit was varying in the range of 0.05–0.11 μg/ml. The relative standard deviations and the accuracy were in the range of 3.1–15.5 and 2.4–16.0%, respectively.
Keywords: 2-Methoxyphenol; 2-Methoxy-4-methylphenol; 2,6-Dimethoxyphenol; 4′-Hydroxy-3′-methoxyacetophenone;