Journal of Chromatography B (v.803, #2)

Update (OFC).

News Section (N1-N2).

Advances in the study of luminescence probes for proteins by Changxia Sun; Jinghe Yang; Lei Li; Xia Wu; Yang Liu; Shufang Liu (173-190).
Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.
Keywords: Reviews; Luminescence probe; Derivatization; Proteins;

The conditions for separation, identification and quantitative determination of epimers 22R and 22S of budesonide by capillary gas chromatography (GC) with FID detection and two various sample injection methods, namely split–splitless and cool on-column, were established. In analysis helium as carrier gas and Rtx®-5 capillary column of 7 m in length along with stationary phase Crossbond® 5% diphenyl–95% dimethyl polysiloxane were used. The individual epimers were identified under specified conditions by using standard samples of different declared concentration of each epimer under investigation: (1) 51.2% of epimer 22R and 47.3% of epimer 22S, and (2) 95.1% of 22R and 4.4% of 22S, as well as Pulmicort®, a preparation containing micronized budesonide as an active substance.It seems that good parameters of preliminary validation achieved by the proposed methods can confirm its suitability for quantitative analysis purpose. The retention times obtained for epimers 22R and 22S, depending on injection technique are about 7.7 and. 8.3 min for split and, approx. 10.3 and 10.9 min for cool on-column. The limits of detection and quantitation are 5.7 and 6.2 ng, for 22R respectively, and 4.3 and 4.8 ng for 22S. The linearity is maintained for concentrations ranging from 0.01 to 0.20 mg/ml. The quantitative analysis features of repeatability, high precision and accuracy confirmed by the obtained results and its statistical evaluation.
Keywords: Budesonide;

Selective and rapid liquid chromatography-mass spectrometry method for the determination of lercanidipine in human plasma by Isam Ismail Salem; Jafer Idrees; Jaafar I Al Tamimi; Pierluigi Farina (201-207).
A specific liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed and validated for the determination of lercanidipine, a dihydropyridine calcium channel blocker, in human plasma. Lercanidipine R-D3 was used as internal standard (IS). The drug was extracted from plasma using liquid–liquid extraction technique utilizing hexane: ethyl acetate as extraction solvent. The samples were analyzed using a prepacked Thermo Hypersil C8 column and a mobile phase composed of a mixture of aqueous acetic acid and triethylamine in methanol. An ion trap mass spectrometer equipped with electrospray ionization (ESI) source operating in the positive ion mode was used to develop and validate the method. The method was proved to be sensitive and specific by testing six different human plasma batches. Linearity was established for the concentration ranges of 0.1–16 ng/ml with a regression factor of 0.9996. The lower limit of quantitation was identifiable and reproducible at 0.1 ng/ml with a precision of 7.2%.
Keywords: Lercanidipine;

A column-switching liquid chromatography–mass spectrometry was developed for quantification of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair. Hair sample was digested in 1N NaOH at 100 °C, and PhIP was extracted using a Blue-Chitin column. The recovery rate was 73%, the limit of quantification was 50 pg/g hair, and intra-day and inter-day variations were 6.3 and 11.7%, respectively. PhIP was found in 42 of the 46 hair samples from 23 healthy volunteers: 110–3878 pg/g hair. The intrapersonal correlation between the first and second analyses was r=0.85 (95% confidence interval, 0.65–0.94). A positive correlation was observed between PhIP levels and melanin content in hair. This study indicates the ability of this method to detect levels of PhIP in hair.
Keywords: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine;

Adsorptive membranes for bilirubin removal by M.E. Avramescu; W.F.C. Sager; Z. Borneman; M. Wessling (215-223).
In this study, we employed ethylene vinyl alcohol (EVAL) adsorptive membranes with bovine serum albumin (BSA) as bioligand for affinity supports for bilirubin (BR) retention. Microfiltration membranes were prepared from ternary or quaternary water/(1-octanol)/DMSO/EVAL systems. To obtain active binding sites for BSA, the EVAL membranes were either chemically functionalized in aqueous and organic medium and by plasma dischargement or physically activated by entrapping of active particles. Static BR removal was determined for all EVAL-BSA membranes. BR retentions relevant for human plasma were gained for the mixed adsorber membranes and additionally investigated in the dynamic mode.
Keywords: Adsorptive membranes; Bilirubin;

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C18 column with methanol–water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r 2>0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91–104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.
Keywords: Skin penetration; Sunscreen agents;

Identification of hydroxy fatty acids by liquid chromatography–atmospheric pressure chemical ionization mass spectroscopy in Euglena gracilis by Lory Z. Santiago-Vázquez; Laura D. Mydlarz; James G. Pavlovich; Robert S. Jacobs (233-236).
Hydroxy fatty acids from Euglena gracilis were identified by reverse-phase high performance liquid chromatography coupled to a mass spectrometer run in atmospheric pressure chemical ionization positive ion mode. These metabolites were converted to methyl esters to improve stability and chromatographic properties. A detection limit of 20 pg/μl per injection was determined for 5-HETE methyl ester based on the signal to noise ratio of the m/z 317 ion which corresponds to the loss of a hydroxyl group (M-17) and the major fragment in all HETE methyl esters studied. This is the first report for these metabolites in E. gracilis.
Keywords: Euglena gracilis; Hydroxyl fatty acids;

Preparation of Concanavalin A-adsorbents by immobilization on Sepharose activated with 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP-reagent) is reported. High immobilization yields of lectin (above 90%) were attained using an optimized CDAP-activating protocol. The effect of ligand density on the performance of the adsorbent for specific binding of glycoproteins was studied using horseradish peroxidase (HRP) as a model. Adsorption yields of pure HRP exceeding 90% were obtained with Con A-derivatives containing not <20 mg of immobilized Con A/ml of packed gel. With lectin content of 2 mg/(ml of packed gel), only 20% of HRP was adsorbed. Purification of peroxidase from horseradish roots extract was successfully accomplished on Con A–Sepharose with high Con A content.
Keywords: Concanavalin A; Peroxidase;

A sensitive method for the determination of Δ9-tetrahydrocannabinol and its metabolites, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid and 11-hydroxy-Δ9-tetrahydrocannabinol, in rat and guinea pig plasma was developed using high-performance liquid chromatographic separation with electrospray ionization mass spectrometry detection and a simple liquid–liquid extraction technique. The mean recoveries for Δ9-tetrahydrocannabinol, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid, and 11-hydroxy-Δ9-tetrahydrocannabinol were 96, 92, and 85%, respectively. The lower limit of quantification (LLOQ) for all three compounds was 5 ng/ml and the limit of detection (LOD) was 2 ng/ml. This assay method utilizes the increased sensitivity and selectivity of mass spectrometric (MS) detection and a simple extraction step for the determination of Δ9-tetrahydrocannabinol and its metabolites in plasma, and thus yields a more efficient pharmacokinetic analysis method than has previously been described.
Keywords: Δ9-Tetrahydrocannabinol; 11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic acid; 11-Hydroxy-Δ9-tetrahydrocannabinol;

Liquid chromatographic method for the simultaneous determination of different lipid-soluble antioxidants in human plasma and low-density lipoproteins by Henar Ortega; José Luis Coperı́as; Patricia Castilla; Diego Gómez-Coronado; Miguel Angel Lasunción (249-255).
We describe a reverse phase HPLC method, employing a simple methanol:water gradient as mobile phase, for the determination of several lipophilic antioxidants, such as retinol, γ-tocopherol, α-tocopherol, lycopene, α-carotene and β-carotene among others, using UV detection. Additionally, this method allows the simultaneous separation of probucol, an hypocholesterolemic drug with antioxidant properties. Retinol acetate and α-tocopherol acetate were added to samples as internal standards. A NovaPack ODS C18, 150×3.9 mm, 0.4 μm column was used and the flow rate was set constant at 1 m/min, which allowed the separation of all the desired antioxidants in a total run time of 35 min. A photodiode array detector was used because of its advantages to study the purity of the peaks, however, any programmable multiwavelength UV/VIS detector could be employed given the good resolution of the peaks. The analytical recoveries of the studied compounds were >96% and the detection limits were: retinol 0.050 μg/ml, γ-tocopherol 0.137 μg/ml, α-tocopherol 0.906 μg/ml, lycopene 0.022 μg/ml, α-carotene 0.008 μg/ml, β-carotene 0.015 μg/ml and probucol 1.503 μg/ml. The intra- and inter-assay coefficients of variation were calculated by using two human plasma samples with different levels of lipophilic antioxidants. The simplicity, rapidity and economy, make this method suitable for the routine measurement of plasma and low-density lipoproteins antioxidants, and may also be used in large scale epidemiological studies. The method has been used to measure antioxidants in samples from patients undergoing treatment with probucol, showing there is a good correlation between the probucol content in LDL and that in total plasma.
Keywords: Antioxidants, lipid-soluble; Low-density lipoproteins;

Profens were converted into diastereomeric (R)-(+)-1-phenylethylamides using ethyl chloroformate and triethylamine in dichloromethane. Gas chromatographic analysis on dual-columns with different polarities provided complete enantioresolution of eight profens, facilitating chiral discrimination based on matching with retention index sets characteristic of each enantiomer. The present method was linear (r≥0.9992) with good precision (0.8–6.0%) and accuracy (−9.3 to 0.003%), allowing detection of trace (R)-profens in optical purity test on four (S)-profen mixture in a single run. And the method allowed simultaneous enantiomeric screening for ibuprofen enantiomers and their chiral metabolites excreted in urine following administration of racemic ibuprofen.
Keywords: Enantiomer separation; Profens; (R)-(+)-1-Phenylethylamides;

Determination of bioactive eicosanoids in brain tissue by a sensitive reversed-phase liquid chromatographic method with fluorescence detection by Hongfei Yue; Kenneth I Strauss; Michael R Borenstein; Mary F Barbe; Luella J Rossi; Susan A Jansen (267-277).
Arachidonic acid (AA) is metabolized to prostaglandins (PGs) via cyclooxygenases (COX) catalysis, and to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DiHETrEs), and hydroxyeicosatetraenoic acids (HETEs) via cytochrome P450 (CYP450) enzymes. A reliable and robust fluorescence based HPLC method for these eicosanoids was developed. A new selective reverse-phase solid phase extraction (SPE) procedure was developed for PG, DiHETrEs, HETE, and EETs of interest from rat cortical brain tissue. The eicosanoids were derivatized with 2-(2,3-naphthalimino)ethyl-trifluoromethanesulphonate (NE-OTf), followed by separation and quantification at high sensitivity using reverse-phase HPLC with fluorescent detection, and further identified via LC/MS. The derivatization was studied and optimized to obtain reproducible reactions. Various PGs, DiHETrEs, HETEs, EETs, and AA were sensitively detected and baseline resolved simultaneously. LC/MS under positive electrospray ionization selected ion monitoring (SIM) mode was developed to further identify the peaks of these eicosanoids in cortical brain tissue. The method was applied in the traumatic brain injured rat brain.
Keywords: Eicosanoids;

Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography by Yazen Alnouti; Catherine A. White; Michael G. Bartlett (279-284).
An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 μm, 150  mm×2  mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 μg/ml for plasma and amniotic fluid and 0.2–50 μg/ml for the placental and fetal tissues.
Keywords: Lamivudine;

Determination of imatinib (Gleevec®) in human plasma by solid-phase extraction–liquid chromatography–ultraviolet absorbance detection by N Widmer; A Béguin; B Rochat; T Buclin; T Kovacsovics; M.A Duchosal; S Leyvraz; A Rosselet; J Biollaz; L.A Decosterd (285-292).
A sensitive HPLC method has been developed for the assay of imatinib in human plasma, by off-line solid-phase extraction followed by HPLC coupled with UV-Diode Array Detection. Plasma (750 μl), with clozapine added as internal standard, is diluted 3+1 with water and subjected to a solid-phase extraction on a C18 cartridge. After matrix components elimination with 2000 μl of water (in two aliquots of 1000 μl), imatinib is eluted with 3×500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 180 μl 50% methanol. A 50 μl volume is injected onto a Nucleosil 100–5 μm C18 AB column. Imatinib is analyzed using a gradient elution program with solvent mixture constituted of methanol and water containing both 0.05% ammonium acetate. Imatinib is detected by UV at 261 nm. The calibration curves are linear between 0.1 and 10 μg/ml. The limit of quantification and detection are 0.05 and 0.01 μg/ml, respectively. The mean absolute recovery of imatinib is 96%. The method is precise with mean inter-day CVs within 1.1–2.4%, and accurate (range of inter-day deviations −0.6 to +0.7%). The method has been validated and is currently being applied in a clinical study assessing the imatinib plasma concentration variability in a population of chronic myeloid leukemia- and gastro-intestinal stromal tumor-patients.
Keywords: Imatinib; Gleevec;

An ultra sensitive method for the direct measurement of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), an antiviral agent for hepatitis B, in human serum using high performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) has been developed. This method involves the addition of [13C]PMEA (contains 5 13C) as internal standard, the purification and enrichment by a MCX solid phase extraction (SPE) cartridge, and quantitative analysis using LC–MS/MS. The MS/MS is selected to monitor the m/z 272→134 and m/z 277→m/z 139 transitions for PMEA and [13C]PMEA, respectively, using negative electrospray ionization. The MS/MS response is linear over a concentration of 0.1–10 ng/ml with a lower limit of quantitation (LLOQ) of 0.1 ng/ml. The mean inter-assay accuracy (%Bias) for quality control (QC) at 0.1, 0.25, 1.0, and 10 ng/ml are 10, 1.6, −0.8, and 0.0%, respectively. The mean inter-assay precision (%CV) for the corresponding QCs is 3.9, 3.8, 5.3, and 3.4%, respectively. The method has been used to determine PMEA concentration in human serum following a single oral administration of a PMEA pro-drug at dose of 10 and 30 mg.
Keywords: 9-(2-Phosphonylmethoxyethyl)adenine;

Plasma d-penicillamine redox state evaluation by capillary electrophoresis with laser-induced fluorescence by Angelo Zinellu; Ciriaco Carru; Salvatore Sotgia; Luca Deiana (299-304).
d-Penicillamine (d-Pen) is a thiol drug used in the treatment of Wilson’s disease, rheumatoid arthritis, metal intoxication and cystinuria. We have recently described a new capillary electrophoresis (CE) method to measure physiological thiols, in which separation of total plasma homocysteine, cysteine, cysteinylglycine, glutathione is achieved using the organic base N-methyl-d-glucamine in the run buffer. In this paper, we present an improvement of our method that allows a baseline separation of total plasma d-Pen from the physiological thiols. Moreover, reduced, free and protein-bound forms of drug are measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulphosalicylic acid (SSA). After derivatization with 5-iodoacetamidofluorescein (5-IAF), samples are separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary (57  cm×75  μm i.d.) using a phosphate/borate run buffer pH 11.4. In these conditions, the migration time of d-Pen is about 7 min and the time required for each analysis is roughly 10 min. The proposed method has been utilized to measure the various forms of the drug in a d-Pen administered Wilson’s disease patient.
Keywords: d-Penicillamine; Thiols;

Liquid chromatographic assay for riluzole in mouse plasma and central nervous system tissues by Milena Colovic; Eleonora Zennaro; Silvio Caccia (305-309).
An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe™ C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100 ng ml−1 or g−1 using 0.1 ml of plasma and about 100 mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).
Keywords: Riluzole;

Determination of derivatized l-alanosine in plasma by liquid chromatography-tandem mass spectrometry by Alex Gantverg; Gary Elliott; Jean Pineault; Roger Demers (311-315).
A sensitive method was developed for quantitation of the cytotoxic antibiotic l-alanosine in human plasma. Alanosine was extracted from plasma by anion-exchange solid phase extraction, derivatized with dansyl chloride and analyzed by liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization in negative mode. Dansylation led to 50-fold improvement of method sensitivity over non-dansylated alanosine with a resulting 20 ng/ml limit of alanosine quantitation in plasma being achieved. The method was validated and applied for clinical studies of alanosine administered to cancer patients.
Keywords: Derivatization, LC; Alanosine;

Simplified method for determination of rosiglitazone in human plasma by Matthew W Hruska; Reginald F Frye (317-320).
Rosiglitazone is a thiazolidinedione antihyperglycemic drug used in the treatment of type 2 diabetes mellitus. Rosiglitazone is extensively metabolized by cytochrome P450 2C8 and so may have some utility as an in vivo probe for this enzyme. A liquid chromatographic method using sensitive fluorescence detection and simplified sample processing involving protein precipitation with acetonitrile was developed. The isocratic mobile phase consisted of 10 mM sodium acetate–acetonitrile (pH 5; 60:40, v/v) and was delivered at a flow rate of 1 ml/min to an Alltima phenyl column (250  mm×4.6  mm, 5 microm). Detection was by fluorescence at (EX/EM) 247/367 for rosiglitazone and 235/310 for the internal standard betaxolol. Intra- and inter-day precision ranged from 3.1 to 8.5% and 2.3 to 5.7%, respectively. No endogenous interference was observed with either rosiglitazone or the internal standard. The assay is simple, economical, precise, and is directly applicable to human pharmacokinetic studies involving single dose rosiglitazone administration.
Keywords: Rosiglitazone;

Stable 3-nitro tyrosine (3-NO2-Tyr), o-, m-, and p-tyrosine isomers induced by oxidation of tyrosine residues in protein were considered important biomarkers for the existence of toxic oxidizing agents peroxynitrite (ONOO) and OH•, which could lead to such diseases as acute lung injury, neurodegenerative disorders, atherosclerosis, cancers and many other diseases. Therefore, development of an accurate, simple and sensitive method to simultaneously detect o-, m-, and p-tyrosine and 3-NO2-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO2-Tyr, due to the strong fluorescence-quenching characteristic of the NO2 group. In this study, we developed a highly sensitive reversed HPLC–UV method, combined with pre-column cloud point extraction (CPE), to simultaneously determine o-, m-, and p-tyrosine and 3-NO2-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl-N-hydroxy-succinimidyl carbomate (AccQ) at 0.20 mol/l borate buffer (pH 8.80) for 30 min at 70 °C, and pre-concentration with surfactant cloud point extraction. The surfactant-rich phase was then diluted with deionized water and injected directly into the to HPLC column for analysis. A C18 column (3.9 mm i.d. × 300 mm) was used for gradient elution separation at 25 °C and the detection wavelength was at 254 nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO2-Tyr were between 5 and 15 nmol/l. The linear range was from 0.05 to ∼100 μmol/l.
Keywords: Tyrosine; 3-Nitrotyrosine;

A simple method for the measurement of the active leflunomide metabolite A77 1726 in human plasma by HPLC is presented. The sample workup was simple, using acetonitrile for protein precipitation. Chromatographic separation of A77 1726 and the internal standard, α-phenylcinnamic acid, was achieved using a C18 column with UV detection at 305 nm. The assay displayed reproducible linearity for A77 1726 with determination coefficients (r 2)>0.997 over the concentration range 0.5–60.0 μg/ml. The reproducibility (%CV) for intra- and inter-day assays of spiked controls was <5%. The limit of quantification was 0.8 μg/ml. The average absolute recovery was approximately 100%. This assay is suitable for the determination of A77 1726 in plasma of patients taking leflunomide, and is simpler to use than other HPLC methods reported previously.
Keywords: Leflunomide; A77 1726;

Concanavalin A chromatography coupled to two-dimensional gel electrophoresis improves protein expression studies of the serum proteome by Ana Marı́a Rodrı́guez-Piñeiro; Daniel Ayude; Francisco Javier Rodrı́guez-Berrocal; Marı́a Páez de la Cadena (337-343).
In the present study, we show a simple method to analyse human serum proteins using Concanavalin A (Con A) chromatography coupled to two-dimensional gel electrophoresis. Serum samples were separated into two fractions, one mainly containing non-glycosylated and O-glycosylated proteins and the other enriched in N-glycosylated proteins. Both fractions were subjected to two-dimensional gel electrophoresis, and the obtained maps were analysed. The method presented here improves the resolution of the serum proteome, increasing the number of visualized spots over two times and allowing the detection of proteins with lower abundance in serum. We have proved the feasibility of the method comparing the N-glycoprotein fraction of serum from donors and colorectal cancer (CRC) patients.
Keywords: Glycosylation; Concanavalin A; Albumin;

Liquid chromatographic method for the determination of uridine in human serum by M Zilly; P Langmann; R Winzer; A Benesic; D Schirmer; U.A Walker; H Klinker (345-351).
To evaluate uridine levels in humans we developed a very sensitive and specific high-performance liquid chromatographic method for the determination of uridine in serum. We use techniques which are available in a standard analytical laboratory. Chromatographic analysis was carried out on a Phenomenex Aqua C18 5 μ 125A column protected by a guard cartridge system. Potassium dihydrogen phosphate buffer–acetonitrile was used as an eluent and oxypurinol as the internal standard. All sample preparation steps were done at 4 °C and the autosampler was cooled down to 4 °C. The calibration curve was linear throughout the calibration range from 0.25 to 100 μmol/l. This method was primarily established to evaluate uridine serum levels in patients with HIV infection since patients on highly active antiretroviral therapy (HAART) might develop metabolic disturbances that could lead to severe and fatal lactic acidosis due to mitochondrial toxicity. It is suggested that a limited or inadequate uridine supply is at least in part responsible for the onset of such deterioration.
Keywords: Uridine;

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 μM, with LOQ/LOD values of 6 and 2 μM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5–10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.
Keywords: Carbamylation; Cyanate; Urea;

Ion chromatographic determination of thyroxine in urine by L Bhavana; V.J Ajimon; S.L Radhika; M Sindhu; C.S.P Iyer (363-366).
Thyroxine (3,5,3′,5′-tetraiodo thyronine) is administered to patients suffering from endemic goiter as also in cases of non-iodine deficient ethiology and hypothyroidism. It is suggested that the uptake of thyroxine can be monitored by assessing the levels of the same in the urine of patients under treatment. For the purpose, a highly sensitive and selective ion chromatographic procedure is developed. The sample of urine is treated with sodium hydroxide and UV irradiated to convert iodine in thyroxine to iodide. Subsequently, iodide is separated on an anion exchanger AS 4A column using 50 mM NaOH as the eluent and determined spectrophotometrically at 226 nm.
Keywords: Thyroxine;

A high-performance liquid chromatographic method using liquid–liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 μl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 μl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5′-thiol of I and protein in the biological sample. After vortex-mixing for 30 s and adding a 50 μl aliquot of the mobile phase containing the internal standard (10 μg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 μl aliquot of the mobile phase. A 50 μl aliquot was injected onto a C18 reversed-phase column. The mobile phases, 50 mM KH2PO4 (pH=2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH2PO4 (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 μg/ml, respectively.
Keywords: 1-(3-Fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione;

Liquid chromatographic determination of amodiaquine in human plasma by Virendra K Dua; N.C Gupta; V.P Sharma; S.K Subbarao (371-374).
A normal-phase high-performance liquid chromatographic method using dichloromethane– methanol–1 M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min−1 on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml−1. Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.
Keywords: Amodiaquine;

Liquid chromatographic–tandem mass spectrometric method for the quantitation of huperzine A in dog plasma by Yingwu Wang; Dafeng Chu; Jingkai Gu; J.Paul Fawcett; Yi Wu; Wanhui Liu (375-378).
A rapid and sensitive LC–MS–MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane–dichloromethane–2-propanol (300:150:15, v/v/v), chromatographed on a C18 column (5 μm, 50  mm×4.6  mm i.d.) with a mobile phase consisting of acetonitrile–methanol–10 mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05–20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration–time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 μg/kg per day for 15 days) of a sustained-release formulation of huperzine A.
Keywords: Huperzine A;