Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.     

Extraction of oxytocin and arginine-vasopressin from serum and plasma for radioimmunoassay and surface-enhanced laser desorption-ionization time-of-flight mass spectrometry

Oxytocin and arginine-vasopressin (AVP) are secreted into the blood in low concentrations. To analyze these peptides, we investigated two common extraction procedures, acetone-ether precipitation and C(18)-SepPak columns. Recovery from both procedures approached 70-80% of the spiked amount, though the SepPak columns were more efficient. C(18)-SepPak columns were used to sequentially separate oxytocin from AVP by eluting oxytocin first with 98% acetone followed by elution of AVP with 80% acetonitrile. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to analyze oxytocin and AVP extracted with C(18)-SepPak columns from an autistic patient's plasma sample. We conclude that C(18)-SepPaks provide more consistent and efficient peptide extraction from serum or plasma that augments both quantitative and qualitative analysis by radioimmunoassay and SELDI-TOF MS.     

Sample preparation for beta-exotoxin determination in Bacillus thuringiensis cultures by reversed-phase high-performance liquid chromatography.

Beta-exotoxin is a nucleotide analogue produced by the entomopathogenic bacterium Bacillus thuringiensis. We have defined two new HPLC procedures for quantification of this exotoxin in culture supernatants of B. thuringiensis grown in poor or rich medium. The sample is prepared either by precipitation in solvent or by solid-phase extraction. Solvent precipitation is achieved treating the sample with acetone and acetonitrile. Solid-phase extraction is performed with a C18 and an anion-exchange cartridge. Reversed-phase HPLC with gradient elution of the prepared samples gives a limit of quantitation of 2 microg/ml for samples prepared by solvent precipitation and of 0.3 microg/ml for samples prepared by solid-phase extraction.     

Quantification of isoflavones in red clover by high-performance liquid chromatography

An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.     

Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrum.

Growth-promoting activity in bovine colostrum has been detected as the capacity to stimulate protein synthesis in L6 myoblasts. By using this assay as a measure of bioactivity, a growth factor has been purified to near homogeneity from centrifuged colostrum by a series of steps including acid extraction, chromatography on sulphopropyl-Sephadex, followed by adsorption to, and elution from, C18 columns using acetonitrile and propan-1-ol gradients. The purified growth factor has a low solubility at neutral and alkaline pH and has an Mr of 7800 by gel-permeation chromatography. Sequence analysis of the first 30 amino acids from the N-terminus indicated complete identity in this region with human insulin-like growth factor-1. Accordingly we conclude that the purified growth factor is bovine insulin-like growth factor-1.     

Analysis of phenolic acids in honeys of different floral origin by solid-phase extraction and high-performance liquid chromatography

The determination of 18 aromatic and arylaliphatic carboxylic acids in honey from different floral origin using solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography (RP-HPLC) is reported. The behaviour of the solutes on SPE cartridges was predicted from preliminary calculations involving the pK(a) constants of the carboxylic groups, the n-octanol:water partition coefficients and the distribution coefficients at different pH values of the conditioning and washing solvents. The proposed SPE isolation and pre-concentration of the acids was achieved on reversed-phase Bond Elut C18 cartridges using an acetonitrile:tetrahydrofuran (1:1, v/v) elution system. RP-HPLC separations were performed on a Spherisorb ODS-2 column using linear gradient elution with a mobile phase composed of 20 mm phosphate buffer (pH 2.92) and methanol, and with UV detection. The reported SPE and RP-HPLC methods were applied to the analysis of 49 authentic honey samples from various floral sources and the results indicate that they may serve with respect to the quantitative control of a number of phenolic acids in plant-derived foods and medicinal plants.     

Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.     

Sensitive determination of tenofovir in human plasma samples using reversed-phase liquid chromatography

A new high-performance liquid chromatography assay was developed for the determination of tenofovir, a nucleotide analogue, in plasma. A solid-liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase containing Na(2)HPO(4) buffer, tetrabutylammonium hydrogen sulfate and acetonitrile for different elution through a C(18) column with UV detection. The method proved to be accurate, precise and linear between 10 and 4000 ng/ml. The method was applied to determine trough levels of tenofovir in 11 HIV-infected patients with virologic failure under multiple antiretroviral therapy. This method was also successfully applied to a pharmacokinetic study in an HIV infected patient with renal failure.     

An evaluation of the use of Sep-Pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.     

Purification of naturally occurring peptides by reversed-phase HPLC

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M(r) < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 A) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Determination of soyasaponins Ba and Bb in human serum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS-MS) has been developed and validated for the determination of soyasaponins Ba and Bb in human serum using glycyrrhizin as internal standard (I.S.). Soyasaponins Ba and Bb were extracted from human serum by liquid-liquid extraction and cleaned up by C(18) solid-phase extraction (SPE), followed by separation on a C(18) reversed-phase column using acetonitrile/water containing 0.025% acetic acid as a mobile phase for gradient elution. Soyasaponins Ba and Bb, and I.S. were ionized by negative ion pneumatically assisted electrospray and detected by HPLC-MS-MS in the multiple-reaction monitoring (MRM) mode using precursor-->product ion combinations at m/z 958-->940, 942-->924 and 822-->351, respectively. The calibration curves were linear (r(2)>0.991) in the concentration range of 0.5-100.0 ng/mL, with lower limits of quantification of 0.5 and 0.3 ng/mL for soyasaponins Ba and Bb, respectively, in human serum. Intra-day and inter-day relative standard deviations (R.S.D.) were less than 7.9 and 11.3%, respectively. The mean recoveries of soyasaponins Ba and Bb ranged from 92 to 101% and from 85 to 94%, respectively.     

Simultaneous determination of triclabendazole and its metabolites in bovine and goat tissues by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous determination of triclabendazole, its main metabolites (triclabendazole sulphone and triclabendazole sulphoxide) and a marker residue (ketotriclabendazole) in bovine and goat muscle, liver, and kidney samples is developed and validated. Analyte extraction from samples is effectively performed using liquid–liquid extraction by acetonitrile. Chromatographic separation is performed on a C₁₈ reversed-phase column with gradient elution. The analytes are detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring. The limits of detection for analytes are found to be 0.25–2.5 μg/kg in muscle tissues and 1–10 μg/kg in liver and kidney tissues, respectively. The recoveries of edible bovine and goat tissues range from 84.9% to 109.5% when spiked at different levels with analytes, with relative standard deviations generally below 12.8%.     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C₁₈ solid-phase extraction cartridge, elution from a Spherisorb C₈ reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8±3.5% and 89.1±6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 μg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

Analysis of secondary metabolites from eschscholtzia californica by high-performance liquid chromatography

A rapid and precise analytical HPLC method has been developed for screening the major benzophenanthridine alkaloids produced by cell cultures of Eschscholtzia califomica, namely, sanguinarine, chelirubine, macarpine, chelerythrine and chelilutine. Separation was achieved on a C18, reversed-phase column with gradient elution using acetonitrile and 50 mM phosphoric acid. Detection was performed by both fluorescence (lambda(ex) 330 nm, lambda(em) 570 nm) and photodiode array, leading to good selectivity and precision in determining peak purity. A simple and quick sample preparation protocol was elaborated involving a methanolic extraction for the measurement of intracellular concentrations of the alkaloids and a solid phase extraction for their quantification in culture medium. Owing to the non-availability of commercially standards, a method for the purification of chelirubine, macar pine and chelilutine by semi-preparative HPLC was developed. Coupled together, the isolation method and the analytical method were highly reliable for screening the alkaloids of interest produced by E. califomica.     

Determination of methotrexate in human urine at nanomolar levels by high-performance liquid chromatography with column switching

An high-performance liquid chromatographic method with column switching for the detection of less than 4 ng of methotrexate in the urine of oncologic nurses is described. Urine samples were purified by solid-phase extraction on silica-bonded phenyl columns, eluting impurities with ethyl acetate. After elution from the column, the analyte was concentrated ten-fold, evaporating the solvent. On a strong anion-exchange column (Nucleosil 100 SB), methotrexate was separated from the remaining interfering substances, was then switched to a reversed-phase column (LiChrospher 100 RP-18e), and finally eluted by a linear gradient in a solvent system consisting of ammonium formate buffer (pH 2.7) and acetonitrile. Absorbance was monitored at 310 nm. This method has proved to be suitable for detecting traces of methotrexate in urine in order to individualize risks and to reduce further the occupational safety hazard for hospital personnel.     

Determination of 4-amino-m-cresol and 5-amino-o-cresol by high performance liquid chromatography and fluorescence derivatization using fluorescamine

For the determination of two oxidation hair dyes, 4-amino-m-cresol (4-AC) and 5-amino-o-cresol (5-AC), a sensitive isocratic high performance liquid chromatography (HPLC) method using the reversed phase mode was developed. The hair dyes were pre-column derivatized with fluorescamine prior to injection. Sensitivity could be improved 10-fold for 4-AC and 50-fold for 5-AC by fluorescence detection compared to UV detection. The limit of detection was 1 ng/injection for 4-AC and 100 pg/injection for 5-AC, respectively. For the determination of both compounds in aqueous biological matrices in order to simulate conditions for penetration studies with pig skin, a solid phase extraction procedure using C18 cartridges and acetonitrile (ACN) for elution could be developed. Average recovery was 83.4% with a coefficient of variation (CV) of 2.64% for intra-day assay and 3.20% for inter-day assay for 5-AC and 2.89% and 3.41% for 4-AC, respectively.     

Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.     

A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate

A sensitive and reproducible HPLC method for quantitative determination of puerarin (PUE) in rat plasma was developed and validated using 4-hydroxybenzaldehyde as an internal standard. The separation of PUE was performed on a CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The method was validated and found to be linear in the range of 80-12,000ng/mL. The limit of quantification was 80ng/mL based on 100μL of plasma. The variations for intra- and inter-day precision were less than 8.3%, and the accuracy values were between 98% and 105.2%. The extraction recoveries were more than 85%. The method was successfully applied in the comparative study of pharmacokinetics of PEGylated puerarin (PEG-PUE) versus PUE in rats. Compared with PUE, PEG-PUE showed a 5.2-fold increase in half-life of PUE and a 4.7-fold increase in mean residence time. In addition, this method was also successfully applied to determine the low plasma concentration of PUE regenerated from PEG-PUE in vitro.