Identification of insulin-like growth factor-II in human seminal and follicular fluids

Antisera raised in rabbits against synthetic insulin-like growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 micrograms/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant amounts of IGF-II.     

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.     

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10 000 pg/ml. Sample extraction (efficiencies >86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantited using post-column UV-photochemical cyclization coupled with fluoremetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.     

A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity

A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low.     

Determination of Salvinorin A in body fluids by high performance liquid chromatography-atmospheric pressure chemical ionization

Salvinorin A was quantitated in human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine by negative ion LC-MS/APCI. The method for Salvinorin A has been fully validated, the LLOQ using FDA guidelines is 2 ng/mL for 0.5 mL plasma samples. The linear range was from 2 to 1000 ng/mL. Several derivatives in the Salvinorin family can also be analyzed by this method; d(3)-Salvinorin A was prepared and used as internal standard. The metabolite Salvinorin B can be semi quantitatively determined. The method has been used to establish that Salvinorin B is the principal metabolite of Salvinorin A ex vivo and to establish the analytical method to study in vivo samples.     

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.     

Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus)

A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography. Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin. The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5. On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000. A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets. Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C₈ column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C₁₈ column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.     

High-performance liquid chromatographic analysis of cytosine arabinoside and metabolites in biological samples

A rapid, non-radioactive method to quantitate therapeutically realistic levels of 1-β- -arabinofuranosylcytosine (Ara-C) and its metabolites would be useful both in the clinic, for monitoring drug levels, and in the laboratory for correlating drug levels with cellular and molecular perturbations. Liquid chromatographic analysis of arabinose-nucleoside analogs in biological samples is complicated by the presence of interfering nucleosides and nucleotides. We report the development of two analytic procedures to measure Ara-C and metabolite levels in biological samples. One method uses a quaternary ammonium type anion-exchange resin to achieve isocratic separation in less than one hour. The second method utilizes a boronate-derivatized polyacrylamide column which binds cis-diols to selectively retain cytosine and uridine, while arabinose compounds are eluted with recovery approaching 100%. The eluted compounds are then easily quantitated on a reversed-phase C₁₅ column. The sensitivity of both procedures was sufficient to obtain pharmacokinetic data on Ara-C and uracil-arabinose levels in serum and urine and on Ara-C triphosphate levels in tumor cells.     

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.     

On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Evaluation of high-performance liquid chromatography for the separation and determination of arsenic species by on-line high-performance liquid chromatographic-hydride generation-atomic absorption spectrometry

An on-line high-performance liquid chromatographic-microwave assisted oxidation-hydride generation-atomic absorption spectrometric (HG-AAS) system (using columns of different kinds) has been developed for the determination of arsenite, arsenate, dimethylarsinate (DMA), monomethylarsonate (MMA), arsenobetaine (AsB) and arsenocholine (AsC) in environmental samples. Ion-pair reversed-phase chromatography using tetrabutylammonium phosphate as the ion-pair reagent and anion-exchange chromatography were evaluated and the analytical performances of each are reported. The detection limits were 97–143 and 10–30 μg l⁻¹ for ion-pair reversed-phase and anion-exchange chromatography, respectively. The Hamilton PRP-X 100 anionic column was proposed for the determination of the six species; AsB can be quantitated independently of AsC by taking the difference between readings at pH 6 and pH 10.7. The proposed methods were applied to water samples and sediments and their potential for future application was demonstrated.     

Direct coupled column separation and determination of the diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine.

A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 microliters) the glucuronides were isolated by complex formation on a terbium(III) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (UV) or femtomole (fluorescence) range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated.     

High-pressure liquid chromatographic method for the simultaneous quantitative analysis of propranolol and 4-hydroxypropranolol in plasma

A simple and rapid high-pressure liquid chromatographic procedure is reported for the simultaneous quantitative determination of propranolol and 4-hydroxypropranolol in plasma. Following an extraction the samples are chromatographed on a reversed-phase column and the components in the column effluent are detected by fluorescence monitoring. Using 1-ml plasma samples propranolol and 4-hydroxypropranolo concentrations at least as low as 1 ng/ml and 5 ng/ml, respectively, can be quantitated. The reproducibility of the method is satisfactory and no interference from endogenous plasma components or other drugs has been observed. A single plasma sample can be analyzed in approximately 20 min.     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

High-performance liquid chromatographic analysis for the determination of miconazole in human plasma using solid-phase extraction

A high-performance liquid chromatographic method for the determination of miconazole in human plasma is described. A solid-phase extraction was performed on an octadecyl (C₁₈) cartridge. Miconazole was eluted with methanol, separated on a reversed-phase column and was measured by ultraviolet detection at 230 nm. The absolute extraction recovery from plasma samples was 85%. The limit of detection was established as 5 ng/ml. The coefficient of variation of the determination of plasma levels by this method over the standard curve concentration range was less than 10%, except with the concentration of 10 ng/ml. The plasma levels of miconazole in twelve healthy volunteers given a 250-mg oral dose of two tablet forms were determined by this method.     

Determination of hydroxyurea in plasma and peritoneal fluid by high-performance liquid chromatography using electrochemical detection

A sensitive method has been developed for the determination of hydroxyurea in plasma and peritoneal fluid using reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Plasma or peritoneal fluid samples were treated with acetonitrile to precipitate proteins then injected to the HPLC. A C₁₈ analytical column was used to separate hydroxyurea from interfering substances in the biological matrix. The mobile phase, consisting of 0.2 M sodium perchlorate–methanol (95:5, v/v) adjusted to pH 5.0, was delivered isocratically at a flow-rate of 1 ml/min and hydroxyurea was detected using a glassy-carbon electrode operating at an applied potential of +800 mV. Hydroxyurea eluted with a retention time of 3 min. The cycle time for analysis is short and the assay precision is acceptable (C.V. plasma=1.4–3.9%, C.V. peritoneal fluid=2.1–9.7%). The method has been validated and is linear from 25 to 400 ng/ml in plasma and 5 to 30 ng/ml in peritoneal fluid. The method has been shown to be applicable for pharmacokinetic studies.     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.