Simultaneous determination of all-trans and 13-cis retinoic acids and their 4-oxo metabolites by adsorption liquid chromatography after solid-phase extraction

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML₃ subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographis (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41–12.44%), reproducibility (C.V. = 9.19–14.73%), accuracy (C.V. = 3.5–11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C₁₈) columns. The mobile phase is hexanedichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.     

Functionalized membrane supports for covalent protein microsequence analysis

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

Trace enrichment of biological folates on solid-phase adsorption cartridges and analysis by high-pressure liquid chromatography

A procedure involving solid-phase adsorption on bonded silica has been developed for trace enrichment and selective recovery of folate monoglutamates from liver tissue. A variety of reverse-phase (ethyl, octyl, octadecyl, phenyl) and anion-exchange (aminopropyl, quaternary amine, primary/secondary amine) cartridges were tested for their potential to adsorb and elute folate monoglutamates from standard solutions (50 nmol each of H4-pteroylglutamic acid (H4PteGlu), 5-CHO-H4PteGlu, 10-CHO-H4PteGlu, PteGlu, and 5-CH3-H4PteGlu). Quantitative recoveries were obtained from aminopropyl (-NH2) and all reverse-phase cartridges. For the analyses of rat liver folates, 20 ml of clear supernatant obtained from 5 g of tissue was treated with conjugase, which released folate monoglutamates from endogenous stores. Folate monoglutamates were then separated from nonfolate material by selective adsorption and recovery from -NH2 extraction cartridges. The procedure also provided a 10-fold concentrate, which allowed direct analysis by HPLC, using C-18 reverse-phase ion-pair columns coupled with uv detection (290 nm). Experiments with standard folates (n = 3) mixed with liver tissue and carried through the extraction, incubation, and trace-enrichment steps showed the following recoveries: 10-CHO-H4PteGlu, 55 +/- 5.0%; H4PteGlu, 80 +/- 5.0%; 5-CHO-H4PteGlu, 123 +/- 12.0%; and 5-CH3-H4PteGlu, 89 +/- 3.0%. Endogenous compositions of liver folates (n = 5) were as follows: 10-CHO-H4PteGlu, 1.03 +/- 0.3 nmol/g (6.7%); H4PteGlu, 5.70 +/- 1.0 (36.4%); 5-CHO-H4Pte Glu, 1.34 +/- 0.4 (8.7%); and 5-CH3-H4PteGlu, 7.34 +/- 1.2 (48.0%). Chromatographic peaks were identified by their retention times and by comparing their spectral profiles (obtained by a diode array detector) with respective pure folates. We found trace enrichment of biological folates on solid-phase extraction cartridges to be rapid and quantitative. The method allowed, for the first time, direct analysis of tissue folates by HPLC/uv methods.     

Purification of Somatostatin from Frog Brain: Coisolation with Retinal Somatostatin-Like Immunoreactivity

Somatostatin-like immunoreactivity (SLI) was purified from frog brain and retina, and the structure of the brain peptide was determined. Frog brain (101 g) and retinal (45 g) tissues were extracted with 3% acetic acid, yielding 9.6 and 0.44 nmol of SLI, respectively. SLI was further purified by chromatography on a somatostatin immunoaffinity column followed by sequential application to reverse-phase C-18 HPLC columns. The brain and retinal peptides, purified roughly 100,000-fold with net yields of 7.5 and 2.3%, respectively, appeared identical in the final steps of purification. The amino acid sequence of brain SLI, as determined by a gas-phase automated Edman degradation technique, was as follows: Ala-Gly-(Cys)-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-(Cys). Our data indicate that despite structural variations in somatostatins of other lower vertebrates, the amino acid sequence of frog brain and, by deduction, retinal SLI is identical to that of somatostatin tetradecapeptide. These findings support the physiological relevance of studies directed at elucidating the neurotransmitter function of somatostatin using the well-established models of frog brain and retina.     

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective R_F values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.     

Isolation and Properties of a New Kallikrein Inhibitor from Tityus serrulatus Venom

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar afinity to both enzymes, showing an IC₅₀ of 14.3 μM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC₅₀ value of 12.6 μM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide K _i = 2.5 μM. The inhibitor was heat resistant and stable at pH values less than 5.     

High-performance liquid chromatography analysis in the synthesis, characterization, and reactions of neoglycopeptides

Reverse-phase HPLC was utilized to study the synthesis, properties, and reactions of the neoglycopeptides formed by reductive lactosylation of the lysine amino groups of a derivative of the immunostimulatory thymic polypeptide thymosin α₁. During the reaction of N^α-formyldesacetylthymosin α₁, a 28-amino acid polypeptide which contains four lysines, with lactose and sodium cyanoborohydride, over 40 intermediates and products were separated by HPLC and 14 of these partially characterized. Increasing levels of lactosylation reduced the elution time of the modified thymosin α₁ derivatives in several HPLC buffer systems investigated. Furthermore, comparisons of mobilities of intact glucosyl- or lactosyl-N^α-formyldesacetylthymosin α₁ showed that the main determinant of elution time was the total number of monosaccharide units per peptide molecule. HPLC analysis was also shown to be a useful tool for studying the susceptibility of neoglycopeptides to proteolytic degradation and will prove an aid to elucidation of the relative reaction rates of the individual thymosin α₁ lysine residues. It is expected that the trends in peptide mobility described will generally apply to the behavior of naturally occurring glycopeptides and glycoproteins in reverse-phase HPLC.     

Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica

A competitive ELISA employing a polyclonal antiserum raised against leucokinin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg of adult houseflies (Musca domestica). Following solid-phase purification, seven HPLC column steps were used to purify 4.8 nmol of leucokinin-immunoreactive material. Sequence analysis and mass spectrometry were consistent with the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly NH2. This peptide was synthesized and co-eluted with the natural peptide on three different HPLC columns. The activities of natural and synthetic muscakinin were identical, with both producing a 4-5 fold increase in fluid secretion by housefly Malpighian tubules at nanomolar concentrations. The presence of a pair of basic residues (Lys-Lys) suggested muscakinin might be processed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH2 being produced by conversion of an N-terminal glutamine to pyroglutamic acid. However, this analog was 1000-fold less active than the intact peptide, comparable to the activity of AK-V which shares the same C-terminal pentapeptide sequence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide (Musca-DP) from the housefly, and the two peptides act synergistically in stimulating fluid secretion. Muscakinin also increased the frequency and amplitude of contractions by housefly hindgut which might further contribute to the excretory process.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Estimation of the Turnover of 3-Methoxytyramine in the Rat Striatum by HPLC with Electrochemical Detection: Implications for the Sequence in the Cerebral Metabolism of Dopamine

Abstract: A highly sensitive method for the determination of 3-methoxytyramine (3-MT) in nervous tissue is described. The method is based on a rapidly performed isolation of 3-MT on small columns of Sephadex G 10, followed by reverse-phase high-performance liquid chromatography in conjunction with a rotating disk electrochemical detector. The detection limit of the assay (0.5–1 pmol/tissue sample) is about 10% of control value for microwave-killed rats. 3-MT as well as dopamine could be quantified in the same chromatographic run. Inhibition of catechol- O -methyl transferase with tropolone resulted in an exponential decline of 3-MT. From this exponential decline a turnover rate for 3-MT of 1.9 nmol/g/h was calculated. In the same group of rats the turnover rate of homovanillic acid was 9.1 nmol/g/h. From these data it is concluded that in the rat striatum about 80% of homovanillic acid is formed from 3,4-dihydroxyphenylacetic acid and 20% from 3-MT.     

Sample preparation for amino acid determination by integrated pulsed amperometric detection in foods

This report describes a new sample preparation method for food which allows a complete separation of carbohydrates and amino acids prior to their analysis by anion-exchange chromatography and integrated pulsed amperometric detection. Food samples with high carbohydrate concentrations are applied to solid-phase extraction columns containing a strong cation-exchange resin. Carbohydrates are recovered initially; retained amino acids are eluted with 0.2 M CaC l(2) subsequently. The carbohydrate and the amino acid fractions are analyzed. The recovery calculated for 21 amino acids was in the range from 84 to 126%. The sample preparation was tested for amino acid concentrations between 4.2 and 84.0 nmol of each amino acid (between 2.1 and 42.0 nmol of cystine) and correlation coefficients between 0.84 and 0.99 were obtained. The capacity of the solid-phase extraction columns employed was up to 3.7 micro mol. Sample preparation was evaluated with four different food samples: sourdough, skim milk, lemon juice, and potato.     

Structure of Somatostatin Isolated from Bovine Retina

Abstract: Somatostatin-like immunoreactivity (SLI) from bovine retina was purified and its structure determined. Retinal tissue (1868 g) extracted with 3% acetic acid yielded 18.6 nmol SLI. This peptide was purified by chromatography on an affinity column made with anti-somatostatin antiserum, a reverse-phase C-18 HPLC column, and three sequential applications on a reverse-phase phenyl HPLC column. The peptide was purified 103,000-fold from the initial extract with an overall yield of 14.4%. Amino acid sequence determination by an automatic Edman degradation technique revealed the sequence to be as follows: Ser - Ala - Asn - Ser - Asn - Pro - Ala - Met - Ala - Pro - Arg - Glu - Arg - Lys - Ala - Gly - (Cys) - Lys - Asn - Phe - Phe - Trp - Lys - Thr - (Phe, Thr, Ser, Cys). The apparent identity of this peptide with somatostatin octacosapeptide (S28) purified from other mammalian tissue indicates the phylogenetic conservation of its structure and facilitates the use of the retina as a model system for studying the neurotransmitter function of somatostatin.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Partial purification of a novel stimulant of gastric acid secretion from canine non-antral mucosa

A novel stimulant of gastric acid secretion was extracted and purified from the non-antral gastric mucosa of the canine stomach and some of its biological properties were examined. Tissue was boiled in water and extracted in 2% trifluoroacetic acid. The stimulatory activity was purified by a combination of reverse-phase high pressure liquid chromatography (HPLC) and gel filtration. Fractions were assayed for a stimulation of basal, pentagastrin- and histamine-stimulated gastric acid secretion in the anaesthetized rat. Stimulatory activity was eluted from reverse-phase HPLC columns with acetonitrile and its elution from Sephadex G-10 and G-50 columns suggested a molecular weight of 1,000 to 3,000. The highly purified extracts enhanced basal, pentagastrin- and histamine-stimulated acid secretion in the rat. A stimulatory fraction was purified which was devoid of immunoreactive gastrin and gastrin-releasing peptide and contained only small amounts of histamine. Its chromatographic properties differed from those of histamine and acetylcholine. On two occasions the stimulant was purified to homogeneity and found to contain amino acids. Insufficient pure material was obtained for full characterization. The stimulant has been tentatively called oxyntin.     

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets

Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C₁₈ reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-¹⁴C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations.     

A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor

We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.     

Divergent solid-phase synthesis and candidacidal activity of MUC7 D1, a 51-residue histidine-rich N-terminal domain of human salivary mucin MUC7.

Domain 1 of the low-molecular-weight human salivary mucin, designated MUC7 D1, spans the 51 N-terminal amino acid residues. This domain contains a 15-residue basic histidine-rich subdomain (R3-Q17) which has 53% sequence similarity to histatin 5 (Hsn-5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1-15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn-5. We were therefore intrigued to explore the candidacidal potency of the full-length MUC7 D1 (51-mer). Linear solid-phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid-phase step-by-step synthesis were circumvented either by double-coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse-phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1-15mer and Hsn-5. The MUC7 D1 candidacidal activity was assessed against azole-sensitive and azole-resistant C. albicans strains and was found, unlike that of the MUC7 D1-15mer, to be comparable with that of Hsn-5, indicating that in addition to Hsn-5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1-15mer, and of Hsn-5, appears to be largely dependent on peptide charge, irrespective of alpha-helical structure.     

Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.