Journal of Chromatography B (v.832, #1)

The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach by Simona Viglio; Laura Annovazzi; Bice Conti; Ida Genta; Paola Perugini; Chiara Zanone; Begoña Casado; Giuseppe Cetta; Paolo Iadarola (1-8).
The aim of the present article is to review the efforts performed in the past two decades by numerous research groups for the development of methods that allow a correct diagnosis of prolidase deficiency (PD), a rare autosomal recessive disorder and for the rationalization of a possible therapeutic intervention on these patients. In particular, the interest of the reader is focused on the application of capillary electrophoresis (i) for the detection of biological markers that reflect the pathological feature of the disease and (ii) for the determination of the efficiency of a carrier system in delivering prolidase inside cells in a possible therapy based on enzyme replacement.
Keywords: Prolidase deficiency; Capillary electrophoresis; Liposomes; Diagnosis; Urine; Imidodipeptides;

To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS).Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6 N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 ×  g for 10 min and the supernatant (10 μL) injected into a Biorad CAT/MET analytical column fitted to the LC–MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines.pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 μL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 μmol/L for urines. Imprecision for spiked serum samples (n  = 10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n  = 10) was between 2.9 and 4.0% for apABG and 6.0–12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n  = 5) are 2.9 ± 2.3 umol/L (mean ± S.D.). This group also had total serum catabolites of 11.9 ± 7.6 nmol/L and serum folate of 35.3 ± 5.8 nmol/L. Serum from patients being tested for PTH (n  = 11) had serum folate levels of 27.0 ± 10.4 nmol/L with total serum catabolites of 20.4 ± 23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n  = 18) were 33.9 ± 22.7 and 11.4 ± 8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n  = 19) was 581.8 ± 368.4 nmol/L.A simple, reliable and highly specific method by LC–MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.
Keywords: pABG; apABG; Folate catabolite; Folate; Tandem mass spectrometry;

Sensitive and specific LC–MS/MS method for the simultaneous measurements of viramidine and ribavirin in human plasma by Yifei Liu; Christine Xu; Rongzi Yan; Charmaine Lim; Li-Tain Yeh; Chin-chung Lin (17-23).
Viramidine is a prodrug of ribavirin. To facilitate pharmacokinetics studies of viramidine in man, a sensitive and specific LC–MS/MS method for the simultaneous analyses of viramidine and ribavirin in human plasma was developed and validated. The method involved the addition of [13C]viramidine and [13C]ribavirin as internal standards, protein precipitation with acetonitrile, HPLC separation, and quantification by MS/MS system using positive electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor → product ion transitions were monitored at 245 → 113, 250 → 113, 244 → 112, and 249 → 112 for ribavirin, [13C]ribavirin, viramidine, and [13C]viramidine, respectively. The calibration curves for viramidine and ribavirin were linear over a concentration range of 1–1000 ng/mL. For both viramidine and ribavirin, the lower limit of quantification (LLOQ) was 1 ng/mL. For viramidine, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated good precision (%CV between 1.0 and 7.0%) and accuracy (%bias between −4.3 and 5.2%). For ribavirin, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated similar precision (%CV between 0.8 and 8.3%) and accuracy (%bias between −5.8 and 9.4%). Both viramidine and ribavirin were stable in human plasma stored at room temperature for at least 3 h, 4 °C for at least 6 h, and for at least three freeze–thaw cycles. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of viramidine and ribavirin in man following administration of viramidine.
Keywords: Viramidine; Ribavirin; Specificity; Sensitivity; Hepatitis C; Measurement;

A sensitive and accurate stable isotope dilution GC/MS assay was developed and validated for the quantification of succinylacetone (SA) in plasma and amniotic fluid (AF). SA is pathognonomic for tyrosinemia type I, a genetic disorder caused by a reduced activity of fumarylacetoacetate hydrolase (FAH). In untreated patients, SA can easily be measured in plasma and urine because the expected concentrations are in the μmol/L range. Due to a founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia type I, hence, the quantification of SA in AF or plasma of treated patients in the nmol/L range becomes very useful. The method utilizes 13C5-SA as an internal standard and a three-step sample treatment consisting of oximation, solvent extraction and TMCS derivatization. The assay was validated by recording the ion intensities of m/z 620 for SA and m/z 625 for ISTD in order to demonstrate the precision of measurements, the linearity of the method, limit of quantification and detection (LOQ and LOD), specificity, accuracy, as well as metabolite stability. Values for the intra-day assays ranged from 0.2 to 3.2% while values for the inter-day assays ranged from 1.9 to 5.6% confirming that the method has good precision. A calibration plot using SA detected by GC/MS gave excellent linearity with a correlation coefficient of 0.999 over the injected concentration range of 5–2000 nmol/L. LOQ and LOD were 3 and 1 nmol/L, respectively. The usefulness of this method was demonstrated by SA quantification in an AF sample of an affected fetus and in plasma of patients treated with NTBC. The results demonstrate that this novel GC/MS method may be a valuable tool for metabolic evaluation and clinical use.
Keywords: Tyrosinemia; GC/MS; Succinylacetone; NTBC;

A sensitive and selective liquid chromatography–tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range 0.05–30 ng/ml with 0.5 ml of plasma using diphenhydramine as the internal standard. Liquid–liquid extraction using a mixture of diethyl ether and dichloromethane was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring (SRM) mode using the atmospheric pressure chemical ionization (APCI) technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitrile–water–formic acid (70:30:0.5), at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intra-day precision (%R.S.D.) were less than 8.5% and accuracy (%error) was less than −2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 20 healthy volunteers.
Keywords: Zolmitriptan; Triptan; Pharmacokinetics; Liquid chromatography–tandem mass spectrometry;

Quantitative determination of 20-hydroxyecdysone in methanolic extract of twigs from Vitex polygama Cham. by Margareth Borges Coutinho Gallo; Flávio Luis Beltrame; Paulo Cezar Vieira; Quezia Bezerra Cass; João Batista Fernandes; Maria Fátima das Graças Fernandes da Silva (36-40).
20-Hydroxyecdysone (20E) is effective in stimulating protein synthesis, therefore, it has been largely used as anabolic agent in several commercial formulas. Phytochemical study of methanolic extract of twigs from Vitex polygama, used in traditional Brazilian medicine as emenagogue, yielded a large quantity of 20E. This finding led us to developing and validating a simple and reliable method to determine 20E in the surveyed extract. Chromatographic separation of 20E was achieved on a phenyl-hexyl-based column using reversed elution mode. Extract was cleaned-up by solid phase extraction employing C18 cartridge, and an absolute recovery of 97% was acquired. External standard and standard addition calibration graphs were obtained and good linearity was accomplished (r  > 0.999 for both curves). The limit of quantification and detection were determined. The results for accuracy fell within the −5 to +7% range.
Keywords: Tarumã; Verbenaceae; Polypodine A; External standard; Standard addition; Solid-phase extraction;

Protein depletion from blood plasma using a volatile buffer by Dmitri Sitnikov; Donovan Chan; Eric Thibaudeau; Marc Pinard; Joanna M. Hunter (41-46).
Removal of high abundance proteins is widely used in sample processing for proteomics studies of blood plasma. Immunoaffinity (IA) depletion is currently the most specific method for performing this step. Historically, IA depletion matrices have been designed to be used with inorganic buffers. However, the presence of salts in depleted samples presents a particular problem, and these must be removed in order to make samples compatible with post-depletion processing. Desalting (dialysis, ultrafiltration, size-exclusion, etc.) usually diminishes sample integrity due to labware associated losses. Moreover, these steps require additional labor, increasing the processing time and cost of analysis. In order to avoid these problems, we have developed an IA method using a volatile buffer that can be removed from depleted samples by lyophilization. This method allows the execution of reproducible and efficient depletion of blood plasma in a semi-automated manner.
Keywords: Immunoaffinity depletion; Volatile buffer; Plasma profiling; Proteomics; Multiple affinity removal system; Albumin; Immunoglobulin G;

An accurate, reproducible and sensitive method for the quantitative determination of latrunculins A and B in the organic extract of the Red Sea sponge Negombata magnifica was developed and validated. Latrunculin A and B concentrations were determined by RP-C18-HPLC and a mobile phase consisting of acetonitrile and water (60:40, v/v). The flow rate utilized was 1 mL min−1 and the detector was set at 235 nm. The HPLC analysis of several N. magnifica samples collected from different locations in the Red Sea revealed that Ras Mohamed had the highest concentrations of latrunculin A, while Safaga had the highest levels of latrunculin B. Also, a comparison between latrunculin concentrations in the summer and winter revealed that the yield of latrunculins were generally higher in the winter.
Keywords: Negombata magnifica; Red Sea; Latrunculin A; Latrunculin B; Season; Location; High performance liquid chromatography;

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 × 10−4  M luminol added to the CE running buffer and 5.0 × 10−4  M NaBrO in 100 mM NaCO3–NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 × 10−6  M (S/N = 3). The precision (R.S.D.) on peak height (at 1 × 10−5  M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at ∼1950 ng/g wet tissue.
Keywords: Capillary electrophoresis; Chemiluminescence detection; Agmatine; Tissue samples; Luminol;

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography–mass spectrometry by Jun Ueyama; Isao Saito; Michihiro Kamijima; Tamie Nakajima; Masahiro Gotoh; Takayoshi Suzuki; Eiji Shibata; Takaaki Kondo; Kenji Takagi; Ken-ichi Miyamoto; Junki Takamatsu; Takaaki Hasegawa; Kenzo Takagi (58-66).
In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography–mass spectrometry (GC–MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 °C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 μg/L for DMP, and 0.1 μg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1–300 μg/L, and the mean recovery was 56.7–60.5% for DMP, 78.5–82.7% for DEP, 88.3–103.9% for DMTP, and 84.2–92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 μg/L for DETP, respectively. The present method, which does not require special equipment except for GC–MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.
Keywords: Organophosphorus insecticide; Urinary metabolite; Dialkylphosphates; GC–MS; Biological monitoring;

Liquid chromatography–tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver by Jérôme Bouligand; Alain Deroussent; Angelo Paci; Jackie Morizet; Gilles Vassal (67-74).
A liquid chromatography/tandem mass spectrometry assay of glutathione (GSH), glutathione disulfide (GSSG) and of precursors (γ-glutamyl-cysteine, cysteinyl-glycine, cysteine, cystine, homocysteine and homocystine) was developed to study glutathione synthesis in mice liver. After iodoacetic acid derivatization, the analytes were analyzed using reversed-phase gradient HPLC and detected using multiple reaction monitoring. Linear calibrations were performed over the concentrations range of 100–10,000 ng/mL for the thiol-containing precursors and extended up to 100,000 ng/mL for GSH and GSSG. The method was validated for each compound with inter-day accuracy below 11.9% and with precision below 15%. The method showed low limits of quantitation of 100 ng/mL for each thiol-containing compound and GSSG and of 200 ng/mL for other disulfides.
Keywords: HPLC–MS/MS; Glutathione; Disulfide glutathione; Glutathione precursors; Liver; Mice;

Six fishermen were victims (including one death) of food poisoning from unknown fish on their boat in central Taiwan Strait, in April 2001. The symptoms were like those of tetrodotoxin (TTX) poisoning. As there was no remaining fish, a new protocol was developed to determine TTX in the urine and blood of the victims. The urine and blood samples were cleansed using a C18 Sep-Pak cartridge column, and the toxin was extracted by methanol. The eluate was filtered through a microcentrifuge filter. The filtrate was freeze-dried, dissolved in distilled water, and determined by LC–MS. The recovery was more than 88.9%. The detection limit was 15.6 nM. A linear relationship between response and concentration was obtained between 93.75 and 9375 nM of TTX. It was shown that the urine and blood of the victims contained TTX. The range of TTX was 4.5–40.6 nM in blood and 47–344 nM in urine. Judging from the symptoms of the victims and the experimental data, the causative agent of the food poisoning was identified as TTX.
Keywords: Tetrodotoxin; Food poisoning; LC–MS;

Determination of drugs of abuse and their metabolites in human plasma by liquid chromatography–mass spectrometry by Marta Concheiro; Ana de Castro; Óscar Quintela; Manuel López-Rivadulla; Angelines Cruz (81-89).
A method, using 0.2 ml of plasma, was designed for the simultaneous determination of morphine, 6-monoacetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, benzoylecgonine and cocaine. The drugs were analysed by LC–MS, after solid phase extraction in the presence of the deuterated analogues. Reversed phase separation on an Atlantis dC18 column was achieved in 10 min, under gradient conditions. The method was full validated, including linearity (2–250 ng/ml, r 2  > 0.99), recovery (>50%), within-day and between-day precision and accuracy (CV and bias <15%), limit of detection (0.5 and 1 ng/ml) and quantitation (2 ng/ml), relative ion intensities and no matrix effect was observed. The procedure showed to be sensitive and specific, and was applied to 156 real cases from road fatalities (7.1% cases positive to cocaine and 0.6% to designer drugs).
Keywords: Drugs of abuse; LC–MS; DUID; Plasma;

An attempt for the simultaneous separation of salbutamol (Sal) and bupivacaine (Bup) enantiomers was performed by capillary elecytrophoresis with a dual mixture of neutral cyclodextrins as chiral selector. The influence on the separation of several parameters such as buffer composition, pH, the concentration ratio of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) to dimethyl-beta-cyclodextrin (DM-β-CD) was investigated. A better separation was obtained for Sal and Bup with the CD mixtures compared to the use of HP-β-CD or DM-β-CD alone. The best simultaneous separation of Sal and Bup enantiomers was achieved with a 20 mM HP-β-CD and 20 mM DM-β-CD at pH 2.5 in a triethanolamine (TEA)/phosphate buffer. This method-utilized chlorphenamine (Chl) as an internal standard was found to be linear in the range 0.5–100 μg/mL and 0.5–150 μg/mL for Sal and Bup enantiomers, respectively. The limits of detection for both enantiomers of Sal and Bup were 0.18 and 0.24 μg/mL, respectively. The proposed method was applied to monitor Sal and Bup enantiomers concentration change in rat blood samples obtained from a male rat after celiac doses administration 0–30 min of Sal and Bup racemate. The method could also be used to determine Sal enantiomers in a pharmaceutical aerosol.
Keywords: Chiral separation; Capillary electrophoresis; Mixtures of neutral cyclodextrin; Basic pharmaceutical; Acrosol;

Quantitative evaluation of sphingolipids using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry with sphingosylphosphorylcholine as an internal standard by Takehisa Fujiwaki; Masaru Tasaka; Nobuyuki Takahashi; Hironori Kobayashi; Yo Murakami; Toshio Shimada; Seiji Yamaguchi (97-102).
Fabry disease is a glycolipid storage disorder caused by a defect of α-galactosidase A, and characterized by the systemic deposition of glycosphingolipids with terminal α-galactosyl moieties, mainly globotriaosylceramide, in tissues. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed the sphingolipids in the cardiac valves from a 49-year-old male patient with Fabry disease who suffered from congestive cardiac failure. Crude lipids were extracted from the cardiac valves with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) ion peaks with m/z values corresponding to different ceramide trihexoside (CTH) species were detected; (b) with sphingosylphosphorylcholine (SPC) as the internal standard for semi-quantification of CTH, the relative peak height of CTH was calculated and plotted versus its amount loaded on the sample plate for MALDI-TOF-MS. The relative peak height of CTH with fatty acid C16:0 showed linearity between 0 and 50 ng CTH (regression coefficient, r  > 0.95); (c) semi-quantitative analysis revealed striking accumulation of CTH in the cardiac valves from the patient with Fabry disease. It was indicated that the accumulation of CTH in cardiac valves from Fabry disease patients can be detected with the DE MALDI-TOF-MS method. SPC is commercially available, and this semi-quantitative method involving MALDI-TOF-MS was found to be convenient, reliable and useful for CTH. It is expected to be applied to the quantification of CTH in small amounts of body fluids or other tissues and to clinical examination. It is also expected to be applicable to the quantification of other glycosphingolipids.
Keywords: Delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Fabry disease; Sphingolipid;

New method for HPLC separation and fluorescence detection of malonaldehyde in normal human plasma by Jianjun Mao; Hongwu Zhang; Jia Luo; Li Li; Rui Zhao; Renen Zhang; Guoquan Liu (103-108).
A new method for the detection of free and total malonaldehyde (MDA) in human plasma samples based on the derivatization of MDA with 9-fluorenylmethoxycarbonyl hydrazine (FMOC-hydrazine) in an acidic medium was developed. Derivatization was achieved after 4 h at 50 °C. The derivatized samples were analyzed by HPLC using a reversed-phase C18 column with fluorescence detection (Ex = 270 nm, Em = 310 nm). The benefit of this direct injection of deproteinized plasma is to avoid the use of an internal standard. The detection limit was 0.1 pmol (4.0 nmol/L). The recovery of MDA spiked in different human plasma samples was 95.3% (n  = 25; R.S.D. 5.1%) for the hydrolysation procedure. The total and free MDA in plasma of 15 healthy male volunteers are 426 ± 29.8 nmol/L and 153 ± 9.6 nmol/L, respectively.
Keywords: Malonaldehyde; 9-Fluorenylmethoxycarbonyl hydrazine; Plasma; Fluorescence detection;

Flavonoids with the quercetin structure are widely distributed throughout the plant kingdom. Some effects such as their anti-oxidative and radical scavenging capacities are broadly discussed in literature. Furthermore, some Hypericum flavonoids show activity in depression-relevant animal model assays. So far, only one study concerning the pharmacokinetic profile of Hypericum perforatum (St. John's wort, SJW) flavonoids has been reported, but no data concerning their bioavailability in the CNS is on-hand. Thus, we developed a method for the quantification of quercetin, tamarixetin and isorhamnetin, both metabolites of quercetin, in very low concentrations in the rat brain in order to investigate the ability of flavonoids to cross the blood–brain barrier. The brain samples for analysis were taken 4 h after feeding an oral dose of an alcoholic SJW extract or pure isoquercitrin. We found the presence of quercetin and isorhamnetin/tamarixetin after feeding a SJW extract at 7 ng/g brain and 35 ng/g brain, respectively. In addition, we examined blood plasma taken from the same rats to correlate plasma and brain levels. The plasma levels were 350 ng/mL for quercetin and 1006 ng/mL for isorhamnetin/tamarixetin after intake of SJW extract.
Keywords: Quercetin; Flavonoids; Hypericum perforatum; St. John's wort; CNS; HPLC;

Enantioselective assay for the determination of perhexiline enantiomers in human plasma by liquid chromatography by Benjamin J. Davies; Megan K. Herbert; Julie A. Culbert; Simon M. Pyke; Janet K. Coller; Andrew A. Somogyi; Robert W. Milne; Benedetta C. Sallustio (114-120).
Effective use of the antianginal agent perhexiline is difficult because saturable metabolism by the polymorphic cytochrome P450 2D6 (CYP2D6) isoform produces elevated plasma perhexiline concentrations that have been associated with serious hepatic and neurological toxicity. Perhexiline is marketed for therapeutic use as a racemate and there is evidence for differences in the disposition of its enantiomers. The current study describes an enantioselective HPLC-fluorescent method utilising pre-column derivatization with (R)-(−)-1-(1-napthyl)ethyl isocyanate. Following derivatization, the enantiomers are resolved on a C18 column with gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of (+)- and (−)-perhexiline in human plasma following clinical doses and demonstrates sufficient sensitivity, accuracy and precision between 0.01 and 2.00 mg/l for each enantiomer, with intra-assay coefficients of variation and bias <20% at 0.01 mg/l and <10% at 2.00 mg/l, and inter-assay coefficients of variation and biases <15% at 0.03 mg/l and <10% at 0.40 and 0.75 mg/l. The application of this method to plasma samples collected from a patient treated with perhexiline revealed that (+)-perhexiline concentrations were higher than (−)-perhexiline concentrations, confirming the stereoselective disposition of perhexiline. The current study describes an enantioselective method that utilises pre-column formation of fluorescent diastereomers that are resolved on a C18 HPLC column using a gradient of methanol and water.
Keywords: Enantiomer separation; HPLC; Perhexiline;

Detection of sulphamethazine residues in cattle and pig hair by HPLC–DAD by M. Gratacós-Cubarsí; M. Castellari; J.A. García-Regueiro (121-126).
An HPLC method with diode array detection (DAD) is proposed for the detection of sulphamethazine (SMZ) residues in pig and cattle hair. Hair samples were extracted under alkaline conditions (NH4OH 0.2 M for calf samples and NaOH 0.1 M for piglet samples) and purified with a dual solid-phase extraction (SPE) cartridge system (reverse phase/strong-cation exchange). Recovery of SMZ in fortified samples varied from 70 to 85%, with a limit of quantification of 0.155 ng/mg. Residues of SMZ (7.2–59.2 ng/mg) were detected both in calf and piglet hairs after a therapeutic treatment with SMZ, while no interfering peak was observed in samples from untreated animals.
Keywords: Sulphamethazine; Hair analysis; Cattle; Pig; HPLC;

Assay of ochratoxin A in grape by high-pressure liquid chromatography coupled on line with an ESI–mass spectrometry by Anna Maria Timperio; Paolo Magro; Gabriele Chilosi; Lello Zolla (127-133).
In this paper, we propose a method for detection of ochratoxin A (OTA) in grapes by using nano-reversed-phase high-performance liquid chromatography–electrospray ionization–mass spectrometry (nano-RP-HPLC–ESI–MS). The method is rapid, highly sensitive and reproducible. OTA is extracted preferably from the entire acinus, rather than must; using chloroform at long incubation time period, lyophilized, resolubilized in acetonitrile (AcCN) and injected onto a reversed phase capillary or analytical column. Capillary columns are the method of choice because it requires a reduced amount of injected sample and consequently the chloroform necessary for OTA extraction, which is a toxic agent. This method gives a detection limit of femtog/ml, without resorting to an immunoaffinity clean-up or concentration, which makes it by far superior to any other method reported. Moreover, by using MS as a detection method it is possible, in the case of a complex matrix, to measure its molecular mass and to confirm the presence of OTA by MS–MS, which cannot be done by fluorescent detection. The method has a high sample extraction throughput (24/h) and has adequate precision (between batch C.V. <8%) and sensitivity (limit of detection (LOD) = 1 pg/g; limits of quantification (LOQ) = 2 pg/g) for OTA measured.
Keywords: Ochratoxin A; Mycotoxin; Grape; Penicillium and Aspergillus; Nano-HPLC; Electrospray ionization mass spectrometry (ESI);

Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine by S.K. George; M.T. Dipu; U.R. Mehra; P. Singh; A.K. Verma; J.S. Ramgaokar (134-137).
An HPLC procedure developed for the rapid and simultaneous determination of purine derivatives (PD) in ruminants’ urine was investigated, since the adoption of a single method for the simultaneous detection of PD and creatinine was not carried out due to elution of polar co-extractives and also due to overlapping of the peaks of allantoin and creatinine. The experimental conditions chosen in the present study avoid the presence of chemically competitive compounds and afford a good separation of the peaks of allantoin and creatinine. The recoveries of the standard compounds added to urine samples were 94–104%. This method can be proposed as a possible reference method for the estimation of allantoin, uric acid and creatinine in cattle urine.
Keywords: Purine derivatives; Allantoin; Uric acid; Creatinine;

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC–UV after solid-phase extraction by S. Colombo; A. Béguin; C. Marzolini; A. Telenti; J. Biollaz; L.A. Decosterd (138-143).
An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV–diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 μl MeOH/H2O 50/50. A 40 μl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 μg/ml, with a lower limit of quantification of 0.125 μg/ml. The mean absolute recovery of TPV is 77.1 ± 4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2–3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.
Keywords: Tipranavir; Protease inhibitors; Therapeutic drug monitoring; Ion pair HPLC; Solid-phase extraction;

A liquid chromatographic–electrospray ionization mass spectrometric technique was developed for direct quantitation of iophenoxic acid (IA) in serum. IA was spiked into canine, feline, bovine, equine, and porcine sera, extracted, and quantified using negative ion monitoring following chromatographic separation on a Luna C18(2) 3 μm (100 mm × 2.1 mm) reversed-phase column. The limit of detection was 25 ng/mL and the limit of quantification was 50 ng/mL. Inter- and intra-assay accuracy (86–113% and 87–115%, respectively) and precision (1.8–7.7%) were calculated. Analysis of serum collected from feral pigs, raccoons, and opossums following ingestion of IA-marked baits confirmed the appropriateness of this method for bait acceptance studies.
Keywords: LC–MS; Negative ion monitoring; Electrospray ionization; Iophenoxic acid; Serum; Bait acceptance;

Phytoestrogens occur in a variety of foods and are thought to offer a protective effect against a number of complex diseases. Due to the diversity of phytoestrogen conjugates formed in the human body, most assays include an enzymatic hydrolysis step prior to analysis. β-Glucuronidase from Helix pomatia, which also contains sulfatase activity, is popular for this task but contains appreciable levels of some phytoestrogens and related compounds, which could affect accurate quantification at low concentrations. Use of solid phase extraction on a polymeric resin has been found to remove the majority of these compounds from the enzyme, without affecting the enzyme activity for almost all of the analytes tested.
Keywords: Phytoestrogens; Isoflavones; Lignans; Helix pomatia; Glucuronidase; Sulfatase; Solid phase extraction; LC/MS/MS;

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography–mass spectrometry by E. Gallardo; M. Barroso; C. Margalho; A. Cruz; D.N. Vieira; M. López-Rivadulla (162-168).
A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography–mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax™/divinylbenzene (CW/DVB 65 μm) and polydimethylsiloxane (PDMS 100 μm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 μL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 μg/mL for blood, and 0.01 to 50 μg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within ±15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.
Keywords: Solid-phase microextraction; Quinalphos; Gas chromatograpy;

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm × 4 mm, 5 μm particles), the mobile phase consisted of acetonitrile −15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Valsartan; Protein precipitation; Pharmacokinetics;

Validation of the measurement of low concentrations of 5-hydroxytryptamine in plasma using high performance liquid chromatography by Wendy Atkinson; Stephen J. Lockhart; Lesley A. Houghton; Brian G. Keevil (173-176).
A sensitive and rapid assay is described for the measurement of low concentrations of 5-hydroxytryptamine (5-HT) present in human platelet-depleted plasma (PDP) using reverse-phase high performance liquid chromatography (HPLC) with fluorimetric detection. With an analysis time of 12 min, this method is particularly useful for large-scale clinical trials investigating small differences in PDP 5-HT concentrations in conditions such as functional gastrointestinal disorders (FGID). The limit of detection and quantification were 1 and 3 nmol/l, respectively, and the calibration curve linear between 1 and 1000 nmol/l. The within-day and between-day precision were 4.3 and <13.6%, respectively.
Keywords: Validation; Plasma; 5-HT; HPLC;