Journal of Chromatography B (v.816, #1-2)

News Section (N1-N2).

Optimisation of the coupled monoclonal antibody density for recombinant hepatitis B virus surface antigen immunopurification by Raúl Hernández; Liuba Plana; Leonardo Gómez; Néstor Expósito; Jorge Valdés; Rolando Páez; Eduardo Martı́nez; Alejandro Beldarraı́n (1-6).
Using immunosorbents based upon cyanogen bromide-Sepharose CL-4B, we have examined different ligand densities in coupling of monoclonal antibody (MAb) to find the best performance, for recombinant hepatitis B surface antigen (rHBsAg) purification. Three replicates of 5 and 15 cycles of densities ranges: 2.17–2.19, 3.18–3.62, 4.06–4.17, and 5.13–5.40 mg/ml (control); or 1.81–2.47, 3.17–3.41, 4.16–4.28, and 5.16–5.19 mg/ml (control), respectively were evaluated in terms of binding capacity, antigen recovery, ligand leakage and purity of antigen, and compared to the control. Adsorption and antigen recovery of immunosorbents manufactured were not different statistically, eventhough increased 8.08 and 9.90% at a range of 3.17–3.41 mg/ml. At this range, efficiency expressed as productivity and MAb saving was optimal. Ligand leakage and purity of antigen showed similar behaviour among all densities. Aspects related to ligand density in antigen immunoaffinity purification are discussed.
Keywords: Monoclonal antibody density; Immunopurification; Recombinant hepatitis B virus surface antigen;

In this paper, a methodology for the determination of three naturally occurring estrogens (estradiol, estrone and estriol) in pregnant women's urine has been described. The procedure included immunoaffinity column (IAC) extraction of 4 mL of urine sample and subsequent analysis of the extraction by micellar electrokinetic chromatography (MEKC). A multi-target polyclonal antibody that has high affinity to three estrogens was produced. Then the IAC was developed by coupling polyclonal antibody to CNBr-activated Sepharose 4B. The IAC showed high affinity for these estrogens. Recoveries of three estrogens from human serum matrix were greater than 92% with R.S.D. less than 4.5%. The final elute of urine sample was diluted with running buffer and then quantitated with MEKC. The experimental results demonstrated that IAC was a useful technique for extraction and concentration of estrogens from biological samples. Three estrogens levels in six pregnant women's urine were measured by both the present method and enzyme-linked immunoadsorbent assay (ELISA). The results of this method have been found to correlate well with those of ELISA.
Keywords: Estrogens; Polyclonal antibody; Immunoaffinity column;

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the metabolites that have been designated as the marker residues for the veterinary drugs, carbadox and olaquindox, respectively, in swine tissue. The method is suitable for use as a confirmatory method under EU National Surveillance Schemes. Porcine liver samples were subjected to protease digestion followed by liquid–liquid extraction. Further clean-up was performed by automated solid phase extraction (SPE) and was followed by a final liquid–liquid extraction step. Analysis was performed using a narrow bore column HPLC coupled to electrospray MS/MS, operated in positive ion mode. MS/MS product ions were monitored at m/z 102 and 75 amu for QCA, m/z 145 and 102 amu for MQCA and at m/z 106 and 152 amu for the d4-QCA and d7-MQCA internal standards, respectively. The method has been validated at 3.0, 10, 50 and 150 μg kg−1 for both metabolites. The method performance characteristics—the decision limit (CCα) and the detection capability (CCβ) have been determined for QCA at 0.4 and 1.2 μg kg−1, respectively, and for MQCA at 0.7 and 3.6 μg kg−1, respectively.
Keywords: Carbadox; Olaquindox; Quinoxaline-2-carboxylic acid; Methyl-3-quinoxaline-2-carboxylic acid;

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm × 4.6 mm, i.d. 3.5 μm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.
Keywords: Hyperforin; Octahydrohyperforin; Tetrahydrohyperforin; Pharmacokinetic; Rodents;

Application of solid phase microextraction to the determination of strychnine in blood by M. Barroso; E. Gallardo; C. Margalho; S. Ávila; E.P. Marques; D.N. Vieira; M. López-Rivadulla (29-34).
A simple and rapid method based on solid phase microextraction (SPME) via direct immersion followed by gas chromatography coupled with electron impact ionization/mass spectrometry (GC/EI-MS) was developed for the determination of strychnine in blood. Papaverine was used as internal standard (I.S.). Two types of fibre coating were tested, 100 μm polydimethylsiloxane and 65 μm Carbowax™/Divinylbenzene, the latter giving higher recoveries of the compound. The main factors affecting the SPME process, such as sample dilution (1:10), adsorption and desorption times (20 and 10 min, respectively), carry-over effect (not observed), pH and salt addition (no modifications on pH or salt concentration) were optimized. The procedure was validated in terms of linearity (r 2  = 0.9992 for concentrations ranging from 0.10 to 5.00 μg/mL), intra and interday precision (0.93 and 4.62%, respectively at 0.50 μg/mL; 3.33 and 8.06%, respectively at 2.50 μg/mL), sensitivity (6.83 and 8.91 ng/mL for LOD and LOQ, respectively) and extraction recovery (0.54 and 0.39% at 0.50 and 2.50 μg/mL, respectively). The developed procedure was found suitable for forensic investigations and was considered a good alternative to the liquid–liquid extraction methods normally used for the determination of this compound in biological media.
Keywords: Solid-phase microextraction; Strychnine;

Indapamide and internal standard (5-chloro-2-methoxy-N-[2-(4-sulphamoylphenyl)ethyl]benzamide) were isolated from plasma by a single step liquid–liquid extraction in t-butyl methyl ether. The chromatographic separation was achieved on a reversed-phase C18 monolithic column with a mobile phase consisting in a methanol/aqueous 0.1% formic acid mixture and a flow rate of 0.8 ml/min, in isocratic conditions, within 11 min. Target compounds were transferred in an ion trap analyzer via an atmospheric pressure electrospray interface (AP-ESI). The mass analyzer was used in a selected reaction monitoring (SRM) mode, in order to enhance on detection selectivity. Whole method produces quantitation limit for indapamide of 1 ng/ml. Method was successfully applied to assess bioequivalence of two sustained release marketed pharmaceutical formulations of indapamide 1.5 mg coated tablets, carried-out in a single/multiple doses, randomized design.
Keywords: Indapamide; Serum; Liquid–liquid extraction; Liquid chromatography; Electrospray tandem mass spectrometric detection; Bioequivalence; Sustained release formulations;

A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K1, phylloquinone, PK and K2, menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7–9.2% for MK-4, 4.9–9.6% for PK and 6.3–19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R 2  = 0.988), PK (R 2  = 0.979) and MK-7 (R 2  = 0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.
Keywords: Vitamin K; Internal standards; Fluorescence detection;

A novel method was developed for the combined determination of carotenoids and retinoids in fish eggs, which incorporates prior analyte isolation using liquid-liquid partitioning to minimize analyte degradation, and fraction analysis using high-performance liquid chromatography–electrospray (positive)–quadrupole mass spectrometry (LC–ESI(+)–MS; SIM or MRM modes). Eggs from Chinook salmon (Oncorhynchus tshawytscha) were used as the model fish egg matrix. The methodology was assessed and validated for β-carotene, lutein, zeaxanthin, and β-cryptoxanthin (molecular ion radicals [M]• +), canthaxanthin and astaxanthin ([M  + Na]+ adducts) and all-trans-retinol ([(M  + H)–H2O]+). Using replicate egg samples (n  = 5) spiked with β-cryptoxanthin and β-carotene before and after extraction, matrix-sourced ESI(+) enhancement was observed as evidenced by comparable %matrix effect and %process efficiency values for β-cryptoxanthin and β-carotene of 114–119%. In aquaculture-raised eggs from adult Chinook salmon astaxanthin, all-trans-retinol, lutein and canthaxanthin were identified and determined at concentrations of 4.12, 1.06, 0.12 and 0.45 μg/g (egg wet weight), respectively. To our knowledge, this is the first report on a method for LC–MS determination of carotenoids and retinoids in a fish egg matrix, and the first carotenoid-specific determination in any fish egg sample.
Keywords: LC–electrospray–tandem (quadrupole) MS; Carotenoids; Retinoids; Astaxanthin; Fish eggs; Chinook salmon;

In this study, high-performance affinity chromatography was used to characterize the binding of carbamazepine to an immobilized human serum albumin (HSA) column. Frontal analysis was first used to determine the association equilibrium constant and binding capacity for carbamazepine on this column at various temperatures. The non-specific binding of carbamazepine within the column was also considered. The results indicated that carbamazepine had a single binding site on HSA with an association equilibrium constant of 5.3 × 103  M−1 at pH 7.4 and 37 °C. This was confirmed through zonal elution self-competition studies. The value of ΔG for this reaction was −5.35 kcal/mol at 37 °C, with an associated change in enthalpy (ΔH) of −6.45 kcal/mol and a change in entropy (ΔS) of −3.56 cal/mol K. The location of this binding region was examined by competitive zonal elution experiments using probe compounds with known sites on HSA. It was found that carbamazepine had direct competition with l-tryptophan, a probe for the indole–benzodiazepine site of HSA, but allosteric interactions with probes for the warfarin, tamoxifen and digitoxin sites. Changes in the pH, ionic strength, and organic modifier content of the mobile phase were used to identify the predominant forces in the carbamazepine–HSA interaction.
Keywords: Carbamazepine; HSA; High-performance affinity chromatography; Frontal analysis; Zonal elution;

Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column by F. Batifoulier; M.-A. Verny; C. Besson; C. Demigné; C. Rémésy (67-72).
A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO4) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90–111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (−67%), brains (−50%), kidneys (−60%), followed by a complete recovery upon repletion.
Keywords: Thiamine; Thiochromes; RP-amide C16 column;

A quantitative method was developed and validated for rapid and sensitive analysis of pravastatin and R-416, the main metabolite of pravastatin, in human plasma. The analytes were extracted from plasma samples by a solid phase extraction method using a Bond Elut® C8. The method involved the use of liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry. A pravastatin analog, R-122798, was used as the internal standard (I.S.). Separation of pravastatin, R-416 and the I.S. was accomplished using a reverse-phase column (C18). The components eluted were ionized by the APCI source (negative ion) and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 0.1 ng/mL (lower limit of quantification, LLOQ) to 100 ng/mL. The intra-assay precisions (coefficient of variation) for the samples at the LLOQ were 1.8% for pravastatin and 1.6% for R-416. The intra-assay accuracy values were 95.8–107.6% for pravastatin, and 92.6–109.0% for R-416, respectively. Precision and accuracy of quality control (QC) samples were determined at concentrations of 0.5, 10 and 80 ng/mL for all analytes. The intra- and inter-assay precision calculated from QC samples were within 10% for pravastatin and within 11% for R-416. The overall recoveries for pravastatin and R-416 were 75.7–82.1% and 68.6–74.3%, respectively. Pravastatin and R-416 were stable in human plasma for 3 weeks at −20 °C in a freezer, up to 6 h at room temperature, and up to 48 h at 6 °C. This assay method was successfully used to evaluate the pravastatin and R-416 levels in healthy volunteers following oral administration of Mevalotin®.
Keywords: Pravastatin; R-416;

A rapid, selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid–liquid extraction, and separated on a C18 column, using acetonitrile–water–10% butylamine–10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20–10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within ±3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.
Keywords: Ibutilide;

New lignan metabolites in rat urine by Annika I. Smeds; Niina M. Saarinen; Patrik C. Eklund; Rainer E. Sjöholm; Sari I. Mäkelä (87-97).
Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography–tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4′-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, α-conidendrin, and α- and β-conidendric acid.
Keywords: 7-Hydroxymatairesinol; Matairesinol; Monodemethylated matairesinol; Secoisolariciresinol; Lariciresinol; Enterolactone; Isohydroxymatairesinol; 4,4′-Dihydroxyenterolactone; 7-Oxoenterolactone; 7-Hydroxysecoisolariciresinol; 7-Oxomatairesinol; α-Conidendrin; α-Conidendric acid; β-Conidendric acid;

High-performance liquid chromatography with ultraviolet detection for therapeutic drug monitoring of everolimus by Sara Baldelli; Stefano Murgia; Simona Merlini; Stefania Zenoni; Norberto Perico; Giuseppe Remuzzi; Dario Cattaneo (99-105).
We developed and validated a high-performance liquid chromatography–ultraviolet (HPLC–UV) method for determining everolimus concentrations in human whole blood. Sample preparation involved a solid-phase extraction after protein precipitation. The separation of everolimus from internal standard (IS) and endogenous components was achieved using an isocratic elution on an octyl column. The method showed a linear relationship between peak height ratios and blood concentrations in the range of 1–200 ng/mL (r 2  = 0.9997). The observed intra- and inter-day assay imprecision had a coefficient of variation (CV) = 12.8%, and inaccuracy was 11.4%. The method was found to be precise, accurate, and sensible making it useful for routine therapeutic monitoring of everolimus.
Keywords: Everolimus; Validation; High-performance liquid chromatography;

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography–mass spectrometry by Nikolai Stephanson; Helen Dahl; Anders Helander; Olof Beck (107-112).
5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography–electrospray ionization mass spectrometry (LC–MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-2H4) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C18 cartridge prior to injection onto a gradient eluted Hypurity C18 reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6–8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n  = 10) and <6.0% (n  = 9), respectively. The new LC–MS method was highly correlated with an established GC–MS method for urinary 5-HTOL (r 2  = 0.99, n  = 70; mean 5-HTOL/GTOL ratio = 1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.
Keywords: Alcohol biomarker; Ethanol; 5-Hydroxytryptophol glucuronide; LC–MS;

The use and limitations of a methanol plug assisted sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method is described. Using naphthalene-2,3-dicarboxaldehyde (NDA)-labeled dopamine as a model compound, this new method was also used in the determination of dopamine in actual urine samples. An inexpensive violet light emitting diode (LED) was used for the light source, because this is suitable for fluorescence excitation. The number of theoretical plates of the analyte was determined to be ∼1 × 105 and ∼2 × 105 by means of MEKC and sweeping-MEKC and this was improved to ∼1 × 106 when the methanol plug assisted mode was applied. In addition, the detection limit of NDA-labeled dopamine was determined to be 9.1 × 10−7 and 1.2 × 10−8  M by means of MEKC and sweeping-MEKC and this was improved to 4.7 × 10−9  M when the methanol plug assisted sweeping-MEKC mode was applied.
Keywords: Methanol plug; Violet light emitting diode; Capillary electrophoresis; Sweeping-MEKC; Dopamine;

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015–5 mg/l for all five NRTIs. The average accuracies for the assay were 92–102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.
Keywords: Nucleoside reverse transcriptase inhibitors; Abacavir; Didanosine; Lamivudine; Stavudine; Zidovudine; HPLC; HIV;

A simple, rapid, reliable, and economic analytical scheme starting with in situ liquid–liquid extraction and asymmetric (or diastereomeric) chemical derivatization (ChD) followed by gas chromatography (GC)-isotope dilution mass spectrometry (MS) is described for the simultaneous determination of d- and l-amphetamine (AP) and methamphetamine (MA) in urine which could have resulted from the administration of various forms of questioned amphetamines or amphetamines-generating drugs. By using l-N-trifluoroacetyl-1-prolyl chloride (l-TPC) as chiral derivatizing agent, resolutions of 2.2 and 2.0 were achieved for the separation of AP and MA enantiomeric pairs, respectively, on an ordinary HP-5MS capillary column. The GC–MS quantitation was carried out in the selected ion monitoring (SIM) mode using m/z 237 and 251 as the quantifier ions for the respective diastereomeric pairs of AP-l-TPC and MA-l-TPC. The calibration curves plotted for the two pairs of analytes stretch with good linearity down to 45 ng/mL, and the limits of detection and quantitation determined were as low as 40 and 45 ng/mL, respectively. Also, a comparative study using 10 real-case urine specimens previously screened as positive for MA administration showed mostly tolerable biases between the two sums (of concentration) of d- and l-MA obtained via an asymmetric l-TPC-ChD approach and via an ordinary pentafluoropropionylation (PFPA-ChD) approach, respectively, as well as between the two sums of d- and l-AP obtained thereupon, thus validating the proposed analytical scheme as a promising forensic protocol for the detailed analysis of enantiomeric amphetamines in urine.
Keywords: Amphetamine; Methamphetamine; Derivatization, GC; Enantiomer separation;

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis by Xiao-Mian Zhou; Shu-Juan Shao; Guan-Dong Xu; Run-Tao Zhong; Da-Yu Liu; Jian-Wu Tang; Yan-Ning Gao; Shu-Jun Cheng; Bing-Cheng Lin (145-151).
The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.
Keywords: Microchip electrophoresis; DNA methylation; Cancer diagnosis;

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of clemastine in human plasma by Viola Horváth; Antal Tolokán; Andrea Egresi; Tímea Pap; George Horvai; Katalin Balogh-Nemes; Imre Klebovich (153-159).
A highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method (HPLC–MS–MS) has been developed to quantitate clemastine in human plasma for the purpose of pharmacokinetic studies. Sample preparation was carried out by liquid–liquid extraction using deuterated clemastine as an internal standard. Chromatographic separation used a C18 reversed phase polymer column giving an extremely fast total run time of 2 min. The method was validated and used for the bioequivalence study of clemastine tablets in healthy male volunteers (n  = 28). The lower limit of detection proved to be 0.01 ng/ml for clemastine.
Keywords: High-performance liquid chromatography-tandem mass spectrometry; Clemastine; Antihistamine;

A simple liquid chromatographic method was developed for the simultaneous determination of flavonoids from Orthosiphon stamineus Benth, namely sinensitin, eupatorin and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, in plasma. Prior to analysis, the flavonoids and the internal standard (naproxen) were extracted from plasma samples using a 1:1 mixture of ethyl acetate and chloroform. The detection and quantification limits for the three flavonoids were similar being 3 and 5 ng/ml, respectively. The within-day and between-day accuracy values, expressed as percentage of true values, for the three flavonoids were between 95 and 107%, while the corresponding precision, expressed as coefficients of variation, for the three flavonoids were less than 14%. In addition, the mean recovery values of the extraction procedure for all the flavonoids were between 92 and 114%. The calibration curves were linear over a concentration range of 5–4000 ng/ml. The present method was applied to analyse plasma samples obtained from a pilot study using rats in which the mean absolute oral bioavailability values for sinensitin, eupatorin and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone was 9.4, 1.0 and 1.5%, respectively.
Keywords: Orthosiphon; Flavonoids; Sinensitin; Eupatorin; 3′-Hydroxy-5,6,7,4′-tetramethoxyflavone; HPLC;

Removal of lipopolysaccharides from protein–lipopolysaccharide complexes by nonflammable solvents by Miao-Fang Lin; Christie Williams; Michael V. Murray; Philip A. Ropp (167-174).
During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein–LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein–LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein–LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein–LPS complexes, while the separation of LPS from protein–LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.
Keywords: Lipopolysaccharides; LPS; Endotoxin; Ion exchange chromatography; Alkanediol; Nonflammable solvent;

Viscose fiber, a regenerated cellulose, was evaluated for using as a novel matrix for high performance affinity chromatography. With a one-step activation with epichlorohydrin, heparin can be readily covalently attached to the matrix. This heparin–viscose fiber material was used for purifying antithrombin III (AT III) from human plasma. The purity of the AT III from this one-step purification is 93% as measured by SDS-PAGE and the protein recovery yield is about 90%. This column is highly specific as described by the dissociation constant of the complex of immobilized heparin and AT III, which was 2.83 × 10−5  mol/L. And more important, this viscose fiber material demonstrated its excellent mechanical property that allows the flow rate to reach up to 900 cm/h or more.
Keywords: Viscose fiber; High performance affinity chromatography; Heparin; Antithrombin III;

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm × 150 mm, 4 μm) protected by a Nova-Pak C18 guard column (3.9 mm ×  20 mm, 4 μm), where 10 mM KH2PO4–methanol–triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 μl mouse plasma. The assay was shown to be linear over a concentration range of 1–500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT.
Keywords: Topotecan; Anti-TPT antibodies; HPLC;

Stanozolol, a synthetic anabolic androgenic steroid, is often abused in sports to enhance performance. Consequently, the anti-doping laboratories daily screen for its metabolites (3′hydroxystanozolol and 4β hydroxystanozolol) in all urines, mainly by GC–MS, after enzymatic hydrolysis and TMS derivatization. A sensitive and specific method by GC–MS3 has been developed for the identification in urine of 3′hydroxystanozolol at trace levels. Full mass spectra and diagnostic ions are presented and a case report is commented. In this case, it was possible to attest the presence of a low concentration of stanozolol metabolite in a sample obtained from a competition test. This would have not been possible with other analytical techniques used in the laboratory. Through this case report, it was also possible to demonstrate the importance of sampling and the difficulties that has to face the laboratory when the pre-analytical step is not correctly performed.
Keywords: Stanozolol; Anabolic steroids; Doping; Urine; GC–MS n ;

An HPLC system using a new, simple and rapid liquid–liquid extraction and high-performance liquid chromatography–diode array detector method (HPLC–DAD) detection was validated to determine tramadol concentration in rabbit plasma. The method described was applied to a pharmacokinetic study of intravenous tramadol injections in rabbits. The extraction with ethylacetate yielded good response. The recovery of tramadol from plasma averaged 90.40%. Serial plasma samples were obtained prior to, during and after completion of the infusion for determination of tramadol concentrations. Tramadol concentrations were measured using reverse-phase high-performance liquid chromatography and pharmacokinetic application with intravenous tramadol in rabbits revealed that tramadol followed one-compartment open model. Maximum plasma concentration (C max) and area under the plasma concentration–time curve (AUC) for tramadol were 14.3 μg mL−1 and 42.2 μg h mL−1, respectively. The method developed was successfully applied to a simple, rapid, specific, sensitive and accurate HPLC method for investigation of the pharmacokinetics of tramadol in rabbit plasma.
Keywords: Tramadol; Pharmacokinetics; HPLC;

High-performance liquid chromatography method for the quantification of rabeprazole in human plasma using solid-phase extraction by N.V.S. Ramakrishna; K.N. Vishwottam; S. Wishu; M. Koteshwara; S. Suresh Kumar (209-214).
A simple, sensitive and selective HPLC method with UV detection (284 nm) was developed and validated for quantitation of rabeprazole in human plasma, the newest addition to the group of proton-pump inhibitors. Following solid-phase extraction using Waters Oasis™ SPE cartridges, the analyte and internal standard (Pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase Waters symmetry® C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 8%. A linear range of 20–1000 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 2.4–7.2% and 2.2–7.3%, respectively. The between- and within-batch bias was −1.7 to 2.6% and −2.6 to 2.1%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.
Keywords: Rabeprazole;

A simple, sensitive and specific HPLC method with UV detection (284 nm) was developed and validated for quantitation of Etoricoxib in human plasma, the newest addition to the group of nonsteroidal anti-inflammatory drugs—a highly selective cyclooxygenase-2 inhibitor. Following a single-step liquid–liquid extraction with diethyl ether/dichloromethane (70/30, v/v), the analyte and internal standard (Zaleplon) were separated using an isocratic mobile phase of water/acetonitrile (58/42, v/v) on reverse phase Waters symmetry® C18 column. The lower limit of quantitation was 5 ng/mL, with a relative standard deviation of less than 20%. A linear range of 5–2500 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 4.1–5.1% and 1.1–2.4%, respectively. The between- and within-batch bias was −3.8–4.7% and −0.6–9.4%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Etoricoxib in plasma was >90%, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive and simple with between-batch precision of <6% and was used in pharmacokinetic studies.
Keywords: Etoricoxib; Liquid–liquid extraction; Quantitation; Human plasma;

In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3 by Tianxiu Qian; Zongwei Cai; Ricky N.S. Wong; Nai Ki Mak; Zhi-Hong Jiang (223-232).
Metabolism of an anti-tumor active component of Panax ginseng, ginsenoside (20R)-Rg3, was studied for better understanding its pharmacokinetics in rat. LC–MS was used to determine Rg3 and its metabolites in rat plasma, urine and feces samples. An average half-life of 18.5 min was obtained after the ginsenoside was intravenously dosed at 5 mg/kg. However, Rg3 was not detected in rat plasma collected after oral administration at 100 mg/kg. Only 0.97–1.15% Rg3 of the dosed amount was determined in feces. Hydrolysis and oxygenated metabolites were detected and identified in feces collected after oral administration by using LC–MS and MS–MS.
Keywords: Ginsenoside Rg3; Rat metabolism; Pharmacokinetics; Metabolite identifications; LC–MS.;

A sensitive method has been developed and validated for the determination of artesunate and its active metabolite dihydroartemisinin (DHA) in human plasma using artemisinin as an internal standard. Solid phase extraction (SPE) using Oasis HLB extraction cartridges was used for sample preparation and analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ion for artesunate, α- and β-DHA was m/z 221 and for artemisinin was m/z 283. Chromatography was carried out using a Synergi Max-RP, 4 μ, 75 mm × 4.6 mm column using glacial acetic acid 0.1%, acetonitrile and methanol mixture (38:46.5:15.5) as a mobile phase delivered at a flow rate of 0.5 mL/min. The retention times of artesunate, α- and β-DHA and artemisinin were 17.4, 11.8, 18.7 and 13.4 min, respectively, with a total run time of 21 min. The assay was linear over the range 1–3000 ng/mL for artesunate and DHA. The analysis of quality control samples for artesunate 50, 300, 1300 and 2600 ng/mL demonstrated excellent precision with relative standard deviation of 14.3, 11.3, 7.5 and 12.1%, respectively (n  = 5). Recoveries at concentration of 50, 300, 1300 and 2600 ng/mL were 75, 94.5, 74.3 and 75.5%, respectively; similar results were obtained for precision and recovery of DHA. This liquid chromatography–mass spectroscopy (LC–MS) method for the determination of artesunate and DHA in human plasma has superior specification for sensitivity, sample throughput and robustness than previous methods and can reliably quantitate concentrations of both (artesunate and DHA) compounds as low as 1 ng/mL.
Keywords: Artesunate; Antiparasitic; Liquid chromatography–mass spectroscopy; Dihydroartemisinin; Antimalarial;

pH-dependent functional activity of P-glycoprotein in limiting intestinal absorption of protic drugs by Manthena V.S. Varma; Mahua Sarkar; Namita Kapoor; Ramesh Panchagnula (243-249).
A simple, specific and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) method with UV absorbance detection was developed and validated for simultaneous determination of quinidine, verapamil and passive permeability markers, in samples obtained from rat intestinal in situ single-pass perfusion studies. Chromatography was carried out on C18 column with mobile phase comprising of acetate buffer (pH 5.0) and methanol in the ratio of 40:60 (v/v) pumped at a flow rate of 0.6 ml/min and UV detection was employed at 230 and 275 nm. The average retention times for hydrochlorthiazide, frusemide, quinidine, propranolol, and verapamil were 4.9, 5.8, 6.9, 8.9 and 11.3 min, respectively. The calibration curves were linear (R 2  > 0.9995) in the selected range for each analyte. The method is specific and sensitive with limit of quantification as 25 ng/ml for quinidine and verapamil. The intra- and inter-day accuracy and precision were found to be good for all the five analytes. The method was found to be reliable in permeability determination and to estimate pH-dependent P-glycoprotein (P-gp)-mediated efflux transport of quinidine. Weak bases quinidine, propranolol and verapamil showed pH-dependent permeability, where quinidine permeability increased by 3.6-fold when the luminal pH was changed from pH 4.5–7.4. Inhibition of P-gp by verapamil (200 μM) indicated that about 68% and only 35% of passive transport of quinidine was attenuated by P-gp-mediated efflux at pH 4.5 and 7.4, respectively. In conclusion, low passive transport rates of weakly basic P-gp substrates at lower pH, may lead to more accessibility of these molecules to P-gp within enterocytes thus resulting in pH-dependent functional activity of P-gp as protic drugs moves along the gastrointestinal tract.
Keywords: P-glycoprotein; Quinidine; pH-dependent; Rat in situ permeability; RP-HPLC;

A sensitive method has been developed for the trace analysis of alkyl alkylphosphonic acids, metabolites of nerve agents, in urine using a benchtop ion trap mass spectrometer. The acids were isolated from urine by simple solid phase extraction and converted to their pentafluorobenzyl esters. An ion trap mass spectrometer in selected reaction monitoring mode provided limits of detection of 0.1 ng/ml for isopropyl, isobutyl, pinacolyl and cyclohexyl methylphosphonic acids and for ethyl ethylphosphonic acid. The detection limit for ethyl methylphosphonic acid was higher (0.5 ng/ml) due to a lower recovery.
Keywords: Nerve agents; Biomarkers; Alkyl alkylphosphonic acids;

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody by Gisele Serpa; Elisabeth Fátima Pires Augusto; Wirla Maria Silva Cunha Tamashiro; Mariana Borçoe Ribeiro; Everson Alves Miranda; Sônia Maria Alves Bueno (259-268).
The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG1 mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me2+-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG1 in the eluate fractions was high when eluted from Zn2+ complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG1 purification, the protein G-Sepharose.
Keywords: Monoclonal antibody; IgG1; Purification; Affinity membrane; IMAC;

We present a fast and reliable on-line clean-up HPLC-method for the simultaneous determination of the five major urinary metabolites of di-(2-ethylhexyl)phthalate (DEHP) namely mono-(2-ethyl-5-carboxypentyl)phthalate (5carboxy-MEPP), mono-[2-(carboxymethyl)hexyl]phthalate (2carboxy-MMHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono-(2-ethylhexyl)phthalate (MEHP). These metabolites represent about 70% of an oral DEHP dose. We for the first time succeeded to reliably quantify 5carboxy-MEPP and to identify 2carboxy-MMHP as major metabolites in native urines of the general population. The analytical procedure consists of an enzymatic hydrolysis, on-line extraction of the analytes from urinary matrix by a restricted access material column (RAM), back-flush transfer onto the analytical column (betasil phenylhexyl), detection by ESI–tandem mass spectrometry and quantification by isotope dilution (limit of detection (LOD) 0.25 μg/l). Median concentrations of a small collective taken from the general population (n  = 19) were 85.5 μg/l (5carboxy-MEPP), 47.5 μg/l (5OH-MEHP), 39.7 μg/l (5oxo-MEHP), 9.8 μg/l (MEHP) and about 37 μg/l (2carboxy-MMHP). The presented method can provide insights into the actual internal burden of the general population and certain risk groups. It will help to further explore the human metabolism of DEHP—an occupational and environmental toxicant of great concern.
Keywords: DEHP; Phthalates; Urine; Plasticizer; Metabolites; Biological monitoring;

Pitfalls in trimethylsilylation of anabolic steroids by Véronique Meunier-Solère; Daniel Maume; François André; Bruno Le Bizec (281-288).
Mixtures such as N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), ammonium iodide and dithioerythreitol (DTE) or MSTFA, trimethyliodosilane and DTE were used for derivatisation of anabolic steroids extracted from 2 g kidney fat and present at ng kg−1 level. They are leading to unexpected products. Their identity and mechanism of formation have been discussed. A new silylation mixture was developed to overcome these pitfalls: N,O-bis-trimethylsilyl-acetamide was used in combination of 2.5% of MSTFA/I2 (1000:10 (v/w)). A single product consisting in ether-TMS and/or enol-TMS derivative was observed for all tested steroids with a stability demonstrated for at least 48 h. Quantitative application was proved even at the low ng kg−1 level in a complex biological matrices, i.e. kidney fat.
Keywords: Gas chromatography–mass spectrometry; Androgens; Trimethylsilylation; Derivatisation; Enolisation;

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binging protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.
Keywords: Capillary affinity electrophoresis; Hyaluronan; Hyaluronan-binding protein;

Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography–mass spectrometry (HPLC–MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor → product ion combinations of m/z 272 → 215, 258 → 201, and 284 → 201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm × 2.0 mm) 5 μm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2–200 and 250–20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the “cross-talk” effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC–MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.
Keywords: Dextromethorphan; Matrix effect; Dextrorphan;

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method for the simultaneous determination of lansoprazole, a proton pump inhibitor and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human plasma. The test compounds were extracted from 1 mL of plasma using diethyl ether–dichloromethane (7:3, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 3.5 mm × 4.6 mm i.d.) for clean-up and column I (C18 STR ODS-II analytical column, 5 μm, 150 mm × 4.6 mm i.d.) for separation. The peak was detected by a ultraviolet detector set at a wavelength of 285 nm, and the total time for a chromatographic separation was ∼25 min. The method was validated for the concentration range from 3 to 5000 ng/mL. Mean recoveries were 74.0% for lansoprazole, 68.3% for 5-hydroxylansoprazole, and 79.4% for lansoprazole sulfone. Intra- and inter-day relative standard derivatives were less than 6.1 and 5.1% for lansoprazole, 5.8 and 5.8% for 5-hydroxylansoprazole, 4.4 and 5.9% for lansoprazole sulfone, respectively, at the different concentration ranges. This method is suitable for use in therapeutic drug monitoring and pharmacokinetic studies, and provides use tool for measuring CYP2C19 activity.
Keywords: Lansoprazole; Metabolite; HPLC; CYP2C19;

Determination of bulleyaconitine A in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry by Weiyu Weng; Huinan Xu; Jianming Huang; Guoquan Wang; Teng Shen; Jianfang Zhang (315-320).
A sensitive and specific high-performance liquid chromatography (HPLC)–electrospray ionization tandem mass spectrometry (MS–MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid–liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor → product ion combinations at m/z 644.6 → 584.3 and 531.2 → 81.6, respectively. Linearity was established for the concentration range of 0.12–6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.
Keywords: Bulleyaconitine A;

Quantification of the bombesin/gastrin releasing peptide antagonist RC-3095 by liquid chromatography–tandem mass spectrometry by Alberto S. Pereira; Luciane DiLeone; Fabiano H. Souza; Sergio Lilla; Marc Richter; Gilberto Schwartsmann; Gilberto De Nucci (321-326).
Bombesin (BN) and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. BN/GRP and their receptors were shown to be critical for the growth of various human malignancies, such as small-cell lung, prostate, ovary, stomach and breast cancers in the human tumor xenograft model. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of a BN/GRP receptor antagonist RC-3095 (D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leuψ(CH2NH)Leu-NH2), in human plasma by liquid chromatography coupled with tandem mass spectrometry. RC-3095 was extracted from 0.2 ml human plasma by protein precipitation using cold acetonitrile (0.4 ml). The method has a chromatographic run of 10 min using a C8 analytical column (150 mm × 4.6 mm i.d.) and the linear calibration curve over the range was linear from 20 to 10000 ng ml−1 (r 2  > 0.994). The between-run precision, based on the relative standard deviation replicate quality controls, was 5.7% (60 ng ml−1), 7.1% (600 ng ml−1) and 6.8% (8000 ng ml−1). The between-run accuracy was ±0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively. The developed procedure allows the quantitative determination of peptide RC-3095 for pharmacokinetics studies in human plasma.
Keywords: RC-3095; Bombesin antagonist; Peptide; Human plasma; Quantification;

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been described for determination of acyclovir in human serum. Since acyclovir is a polar compound and soluble in aqueous medium and practically insoluble in most of organic solvents, its analysis in biological fluids in currently published HPLC methods, involve pre-treatment of acyclovir plasma sample including deproteinization or solid phase extraction. In present method liquid–liquid extraction of acyclovir and internal standard (vanillin) is achieved using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent. Analysis was carried out on ODS column using methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/L) and triethylamine (2 mL/L, v/v) as mobile phase (pH = 2.3; 5:95, v/v) at flow rate of 2 ml/min. The method was shown to be selective and linear into the concentration range of 10–2560 ng/mL. Accuracy and precision of the method were also studied. The limit of quantitation was evaluated to be 10 ng/mL. This method was applied in bioequivalence study of two different acyclovir preparations after administration of 400 mg in 12 healthy volunteers.
Keywords: Reverse phase chromatography; HPLC; Acyclovir; Serum; Bioequivalence study;

A simple and versatile low-capacity cation-exchange chromatography system for the simultaneous determination of creatinine and UV-absorbing amino acids was developed. The separation column was packed with a newly developed low-capacity sulfoacylated macro-porous polystyrene-divinylbenzene resin selective for amino-acid cations. Urinary creatinine, creatine, tyrosine, histidine, phenylalanine, and tryptophan were simultaneously separated and determined by an isocratic elution with phosphate/acetonitrile eluent in 25 min. Relative standard deviations (R.S.D.) of the retention times for the analytes were between 0.28 and 1.06%. R.S.D. of peak area responses for the analytes were between 0.75 and 3.51%. The r 2 values for the calibration lines were between 0.9994 and 0.9999. The method could provide the creatinine ratios for the analytes, and was applicable to the screening and/or chemical diagnosis of several inherited disorders of amino-acid metabolism such as phenylketonuria (PKU).
Keywords: Creatinine; Amino acids; Inborn errors;

Development and validation of an HPLC-UV method for determination of iohexol in human plasma by Rohit S. Soman; Hamim Zahir; Fatemeh Akhlaghi (339-343).
An HPLC-UV analytical method for estimation of iohexol in human plasma was developed and validated. Protein precipitation and iohexol extraction from plasma (100 μl) was carried out by adding 800 μl perchloric acid (5%, v/v in water) containing iohexol related compound B as the internal standard followed by vortex mixing and centrifugation. The supernatant (90 μl) was then injected onto a μBondapak C18 column (150 mm × 3.9 mm, 10 μm) maintained at 30 °C. The mobile phase comprised of various proportions of acetonitrile and water with a total run time of 12 min and the wavelength of the UV detector was set at 254 nm. The extraction recovery of iohexol from plasma was >95% and the calibration curve was linear (r 2  = 0.99) over iohexol concentrations ranging from 10 to 750 μg/ml (n  = 8). The method had an accuracy of >92% and intra- and inter-day CV of <3.7% and <3.6%, respectively. The method reported is simple, reliable, precise, accurate and has the capability of being used for determination of iohexol in clinical settings.
Keywords: Iohexol; GFR;

Author Index (345-347).

Keyword Index (349-355).