Journal of Chromatography B (v.788, #1)

News section (N1-N2).

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with α-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm×4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.
Keywords: Phenylalanine; Tyrosine;

New approach for the detection of BSH and its metabolites using capillary electrophoresis and electrospray ionization mass spectrometry by Pier Luigi Mauri; Fabrizio Basilico; Pier Giorgio Pietta; Erica Pasini; Diego Monti; Wolfgang Sauerwein (9-16).
Boron neutron capture therapy is a promising binary treatment for cancer. It is based on the nuclear fission that occurs when non-radioactive 10B absorbs thermal neutrons. One of the two boron compounds currently used in clinical trials for this therapy is BSH. To ensure differentiated retention in the tumour versus normal tissue prior to treatment, routine analytical methods to determine pharmacokinetics must be available. For this purpose we have developed a new, easy and time saving approach, in which the separation of boron derivatives is performed by means of capillary electrophoresis (CE). The CE method allows analyses to be performed in short times (less than 18 min), sensitively (LOD 8 pg loaded on the capillary) quantitatively (LOQ 5 μg/ml) and with a high efficiency of separation. Moreover it is simpler than HPLC and more reproducible (intra- and inter-day values were ±1% and ±3%, respectively), and does not require a specific column of derivatization. Mass spectrometry analysis of boron derivatives in different samples was also performed to ensure correct attribution of the CE peaks.
Keywords: BSH; Boron;

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of α-naphthylisothiocyanate (1-NITC) and two metabolites α-naphthylamine (1-NA) and α-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C18 5-μm column, a mobile phase of acetonitrile–water (ACN–H2O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95–106 and 97–103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at −20 and −80 °C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 °C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague–Dawley rat.
Keywords: α-Naphthylisothiocyanate; α-Naphthylamine; α-Naphthylisocyanate;

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS–MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid–liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H]+ ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 μg kg−1 in liver and 5, 15 and 50 μg kg−1 in eggs. The new analytical limits, CCα and CCβ were calculated for liver and were 35.4 and 43.6 μg kg−1, respectively.
Keywords: Halofuginone;

Liquid chromatographic method for the determination of rosiglitazone in human plasma by B.L Kolte; B.B Raut; A.A Deo; M.A Bagool; D.B Shinde (37-44).
A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid–liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C18, 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5–1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.
Keywords: Rosiglitazone;

A sensitive and specific LC/MS/MS method has been developed and validated for determination of ragaglitazar (NNC 61-0029 or DRF 2725) in human plasma. After solid-phase extraction (SPEC® PLUS™ C8) of plasma, separation was performed on a Symmetry Shield™ RP8 column (mobile phase: acetonitrile: 10 mM ammonium acetate, pH 5.6 (40:60 v/v)). Two ranges were validated having LLOQs of either 0.500 or 100 ng/ml and linearity up to either 500 or 50 000 ng/ml. The intra-assay precision and accuracy were 1.1% to 15.7% and 85.8% to 118.2% (range 0.500–500 ng/ml) and 2.0% to 8.8% and 92.9% to 104.8% (range 100–50 000 ng/ml). The method was applied for determination of ragaglitazar in plasma from phase 1 and 2 clinical studies.
Keywords: Ragaglitazar;

A simple, rapid and sensitive HPLC method for the determination of diclofenac in synovial fluid is described. Special attention was paid to the procedure of sample preparation since gel formation may sometimes occur in synovial samples. With a one-step extraction procedure good and reproducible recovery of diclofenac was obtained. A subsequent HPLC assay was adjusted so as to achieve adequate sensitivity and precision needed for analysis of true samples. The results obtained by the described procedure proved the method to be suitable for monitoring concentrations of diclofenac in synovial fluid.
Keywords: Diclofenac; Synovial fluid;

Irinotecan (CPT-11), a camptothecin analog, is metabolized to SN-38, an active topoisomerase I inhibitor, and inactive metabolites, including APC and SN-38 glucuronide (SN-38G). A high-performance liquid chromatographic assay method to simultaneously measure the lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in human plasma was developed. Chromatography was accomplished with a reversed-phase C8 column and fluorescence detection. A gradient mobile phase system was used. The buffer for mobile phase A consisted of 0.75 M ammonium acetate, 5 mM tetrabutylammonium phosphate (pH 6.0), and acetonitrile (86:14, v/v). The buffer for mobile phase B was identical to mobile phase A with the exception of the concentration (50:50, v/v). Precipitation of plasma proteins was performed with cold methanol. The linear range of detection of the lactone and carboxylate forms of SN-38, SN-38G, and APC was 2–25 ng/ml, and 5–300 ng/ml for CPT-11. The limit of quantitation for the analytes ranged from 0.5 to 5 ng/ml. Analysis of patients’ plasma samples obtained before and after CPT-11 administration showed that the assay is suitable for measuring lactone and carboxylate forms of CPT-11, SN-38, SN-38G, and APC in clinical studies.
Keywords: Irinotecan; SN-38;

We have developed a separation system for N ε-(carboxyethyl)lysine (CEL) and N ε-(carboxymethyl)lysine (CML) by HPLC equipped with a styrene–divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N α-acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.
Keywords: N ε-(Carboxyethyl)lysine; N ε-(Carboxymethyl)lysine; Proteins; Aldehydes;

Analysis of acyl-lysophosphatidic acids (LPAs) has clinical importance as a potential biomarker for ovarian and other gynecological cancers or obesity from the point of view of prevention. Here we report a simple sample preparation and analytical method with high sensitivity and specificity for the early detection of gynecological cancers to improve the overall outcome of this disease. We established a novel quantification method for acyl-LPAs in plasma by electrospray negative ionization tandem mass spectrometry (MS–MS) using multiple reaction monitoring mode without conventional TLC step. Protein-bound lipids, acyl-LPAs in plasma were extracted with methanol/chloroform (2:1) containing LPA C14:0 as internal standard under acidic conditions. Following back-extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol and then analyzed. Using ESI-MS–MS with negative ionization MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H] of C16:0, C18:2, C18:1, C18:0 and C20:4 LPA, respectively. Q2 ions selected for MRM was m/z 79, phosphoryl product. Using MS–MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. This method allowed simultaneous detection and quantification of different species of LPAs in plasma over a linear dynamic range of 0.01–25 μmol/l. The method detection limit was 0.3 pmol/ml with correlation coefficient of 0.9983 in most LPAs analyzed. When applied to plasma from normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.
Keywords: Acyl-lysophosphatidic acid;

An efficient, sensitive and rapid analysis of the amino acid neurotransmitters in the cerebral cortex of rats was developed by capillary electrophoresis with laser-induced fluorescence detection and fluorescein isothiocyanate (FITC) derivatization. This method was used to investigate the pharmacological effect of baicalin during cerebral ischemia. Different parameters which influenced derivatization and separation were optimized. The separation of amino acids was carried out in an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 15 mM borate at pH 9.2 and an applied voltage of 17.5 kV. The detection limits for six amino acids were in the range of 2.1×10−11–6.3×10−10 M. The changes in the level of amino acid neurotransmitters in brain cortex of three experimental rat groups were studied by this capillary electrophoresis–laser-induced fluorescence detection method. The results show that cerebral ischemia can cause a significant elevation in the concentrations of Glu, Asp, GABA, and Gly in cerebral cortex. Baicalin administration can attenuate the elevations of Glu and Asp induced by cerebral ischemia. This research demonstrates that baicalin may act as a neuroprotectant during cerebral ischemia.
Keywords: Amino acid neurotransmitters; Baicalin;

Cell extraction and further sample preparation for nucleotide pool analysis using capillary electrophoresis was faster and simpler using volatile extraction solvents (e.g. organic solvents and de-ionized water) compared to the commonly applied acids dissolved in water (e.g. perchloric acid and trichloracetic acid). Temperature had to be controlled during the whole sample preparation process to prevent degradation, and extracts had to be cleaned from proteins and other large molecules prior to capillary electrophoretic analysis to improve reproducibility. Capillary electrophoresis using borate and cyclodextrins in the background electrolyte was used for determining 11 cellular nucleotides simultaneously. In order to optimize the assay, 0–100% acetonitrile, 0–100% ethanol, and 0–100% methanol in de-ionized water were applied to extract nucleotides from mouse lymphoma cells, and nucleotide yields, recovery, and reproducibility were compared. The assay met the commonly accepted validation limits for biological fluids, if 20–80% acetonitrile in water and 40–60% ethanol in water were used as extraction solvents.
Keywords: Nucleotides;

Epidermal growth factor (EGF) is a protein that belongs to the family of growth factors that bind the ErbB receptors, which play a prominent role in the development of carcinomas. We had demonstrated that potato carboxypeptidase inhibitor (PCI) acts as an EGF antagonist. Because of the low affinity of PCI for the epidermal growth factor receptor, it was decided to design EGF mutants with PCI abilities. In order to achieve this we have first cloned, expressed and purified the native protein, EGF. Different expression systems with different locations of the recombinant protein were designed and a purification protocol was designed with those which allowed expression of EGF. Finally, the sample needed folding. Differences in the amount of EGF obtained and its activity were observed depending on the expression system used.
Keywords: Epidermal growth factor;

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma by Nina Isoherranen; Boris Yagen; Ofer Spiegelstein; Amir Steinman; Richard H Finnell; Meir Bialer (125-136).
Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C18 cartridges. The urine samples were prepared by liquid–liquid extraction. The sample volume used was 100 μl of dog plasma, 50 μl of mouse plasma and 20 μl of dog or mouse urine. The quantification range of the method was 1.5–50 mg/l in dog plasma (VTD only), 2.5–250 mg/l in mouse plasma (0.7–90 pmol injected) and 0.04–2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15–20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.
Keywords: Valproyl taurinamide;

Determination of dexamethasone in urine by gas chromatography with negative chemical ionization mass spectrometry by Olga Huetos Hidalgo; Manuel Jiménez López; Elisa Ajenjo Carazo; Manuel San Andrés Larrea; Thea B.A Reuvers (137-146).
Dexamethasone, as some other synthetic corticosteroids, is licensed for therapy in veterinary practice, but its misuse as a growth promotor, often in combination with beta-agonists, is forbidden. In this report an analytical method is described for the detection and confirmation of very low concentrations of dexamethasone in urine. The influence of enzymatic hydrolysis time of samples with glucuronidase was studied. The proposed method consisted of the enzymatic hydrolysis of urine samples, which were then extracted and concentrated using solid-phase cartridges with mixed reversed-phase materials (OASIS). No further clean-up step was found to be necessary. Eluates were derivatized following a previously described method [Analyst 119 (1994) 2557]. Detection, identification and quantification of residues of this compound was carried out by gas chromatography with mass spectrometry in the negative chemical ionization mode. The proposed procedure permits the determination of dexamethasone in urine at levels as low as 0.2 ng ml−1
Keywords: Dexamethasone;

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250×4-mm, 5-μm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)–CH3OH 78–22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV–Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-μl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/μl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/μl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1–8 ng/μl.
Keywords: Cephalosporins; Cefadroxil; Cefaclor; Cefotaxime; Cephalexin;

Cromolyn sodium is a safe compound with potent anti-allergic properties when used locally or topically. Clinical data from systemic exposure is not available because of the poor GI absorption when given orally. In order to evaluate a new approach to enhance the absorption and bioavailability of cromolyn sodium, a sensitive assay was needed to support an oral-dose study in humans. This paper describes a liquid chromatographic−tandem mass spectrometric (LC–MS–MS) method for the analysis of cromolyn sodium in human plasma. The method consists of a two-step extraction with subsequent analysis using a high-performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C18 column followed by a backflush. The total run time is 6 min. The standard curve of cromolyn sodium was over the range of 0.313 to 750 ng/mL with a lower limit of quantitation (LLOQ) of 0.313 ng/mL when 0.5 mL of plasma was used for analysis. The percent coefficient of variation (C.V.) for accuracy and precision (inter-assay and intra-assay) was less than 15% over the validated concentration range and the coefficients of determination, r 2, were >0.991577. The method is simple, sensitive, and selective, and has been successfully utilized for oral cromolyn sodium clinical studies.
Keywords: Cromolyn sodium;

A method for the simultaneous determination of sulfadiazine and trimethoprim in plasma from Beagle dogs was developed and validated. Samples were deproteinized with acetonitrile and extracted with ethyl acetate. Sulfachloropyridazine and ormethoprim were used as internal standards for the sulfadiazine and trimethoprim analysis, respectively. The chromatography was carried out both on an LC–UV (liquid chromatography–ultraviolet detection) and ion-trap LC–MSn (liquid chromatography–mass spectrometric detection) instrument, operating in the positive APCI mode (atmospheric pressure chemical ionization). The purpose of this work was to compare the quantification results of both methods. Both the LC–UV and LC–MS–MS methods were validated for their linearity, accuracy, precision, limit of detection and limit of quantification, according to the requirements defined by the European Community. Calibration curves using plasma fortified between 0.1 and 1 μg/ml of sulfadiazine, 0.1 and 2 μg/ml of trimethoprim, 1 and 20 μg/ml of sulfadiazine showed a good linear correlation (r≥0.9990, goodness-of-fit≤8.4%). The results for the accuracy and precision at 1 μg/ml of sulfadiazine and trimethoprim and at 20 μg/ml of sulfadiazine fell within the ranges specified. The limits of quantification of both methods were 0.1 μg/ml. The limits of detection were 0.019 μg/ml of sulfadiazine and 0.024 μg/ml of trimethoprim for the LC–UV method, and 0.020 μg/ml of sulfadiazine and 0.062 μg/ml of trimethoprim for the LC–MS–MS method. The methods have been successfully applied in a pharmacokinetic study to determine the drug concentrations in plasma samples from dogs. A good correlation between the results of both methods was observed (R=0.9724, slope=1.0239, intercept=−0.2080 μg/ml for sulfadiazine and R=0.9357, slope=1.0433, intercept=0.0325 μg/ml for trimethoprim). The precision of both methods was also tested on the results of the same samples using an F-test (α=0.05), indicating that both methods did not differ in precision.
Keywords: Sulfadiazine; Trimethoprim;

For toxicological purposes, an HPLC assay was developed for the simultaneous determination of haloperidol and atypical antipsychotics (risperidone, 9-hydroxyrisperidone, olanzapine, clozapine) in human plasma. After a double-step liquid–liquid extraction, compounds were separated on a C8 column eluted with a gradient of acetonitrile and phosphate buffer 50 mM pH 3.8. A sequential ultraviolet detection was used (260, 280 and 240 nm). Calibration curves were linear in the range 10–1000 ng/ml. The limits of quantification were 5 ng/ml for all drugs. Average accuracy at four concentrations ranged from 93 to 109%. Both inter- and intra-day variation coefficients were lower than 11% for all drugs. This simple and rapid method (run time<15 min) is currently used for poison management.
Keywords: Haloperidol; Risperidone; 9-Hydroxyrisperidone; Olanzapine; Clozapine;

Measurements of sub-nanomolar concentrations of unmetabolised folic acid in serum by Mary Rose Sweeney; Joseph McPartlin; Donald George Weir; John Martin Scott (187-191).
We describe a combined HPLC/microbiological assay procedure for the sub-nanomolar analysis of unmetabolised folic acid (pteroylglutamate) in human serum. This metabolically unaltered form of the vitamin arises following the consumption of folic acid either in supplemental form or in fortified foods. Following HPLC separation of folic acid from other folate derivatives the folic acid fraction was concentrated by C18 Sep-Pak cartridges and assayed by Lactobacillus casei microbiological assay. The present assay allows the quantitation and kinetic analysis of the effects of consumption of folic acid.
Keywords: Folic acid;

A modified microbore high-performance liquid chromatography-immobilized enzyme reactor-electrochemical detection system for acetylcholine (ACh) and choline (Ch) was developed. The system used the horseradish peroxidase and a solution mediator ferrocene to convert the analyte into an oxidized ferrocene species which was detected electrochemically by reduction at 0 mV. There was an excellent linear relationship between the concentration of ACh/Ch and the peak height over the range of 1–5000 nmol/l. The limit of detection for ACh was 2 fmol/5 μl (S/N=3:1). Compared with the common method recommended by Bioanalytical System Inc. (BAS), this method exhibits a 200-fold improvement in the detection limit. The ACh and Ch levels in rat brain microdialysate were examined.
Keywords: Acetylcholine; Choline; Horseradish peroxidase;

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C8 column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0–100.0 ng/ml for serum, and 50.0–500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.
Keywords: Indapamide;