Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Purification of growth hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C₁₈ cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.     

Evaluation and separation of steroid-bovine serum albumin conjugates by high-performance liquid chromatography

The conventional methods for characterization of steroid immunogens are based on the determination of the total amount of hapten bound to the protein carrier either by the UV spectroscopy or titration of unsubstituted amino groups. These methods do not allow more detailed insight into the immunogen composition. HPLC of the immunogen combined with UV detection is a relatively rapid and convenient method enabling determination of the hapten content in each fraction and, eventually, separation of individual fractions differing in the hapten content or purification of crude product.     

Analysis of pancreatic peptide hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) is examined as a method for separating pancreatic peptides. The method was based on gradient elution with acetonitrile in an acid phosphate buffer (pH 3.10). Apart from human and porcine insulin all the other peptide standards tested (thyrotropin-releasing factor, vaso-active intestinal polypeptide, human C-peptide, porcine C-peptide, somatostatin, porcine glucagon, porcine proinsulin and porcine pancreatic polypeptide) could be separated simultaneously in 40 minutes with a binary gradient composed of five linear segments and increasing from 0 to 60% acetonitrile. Human and porcine insulin could be almost completely resolved by a minimal reduction in the steepness of the acetonitrile gradient. Repeated injections of human C-peptide and porcine insulin resulted in a coefficient of variation of less than 1.5% in the retention times. The use of 125I-labelled peptides gave recoveries exceeding 90%. HPLC of acid ethanol extracts of autopsy pancreases from three infants showed that the immunoreactivity of the peptides measured remained unaffected by the chromatography. Both immunoreactive C-peptide and immunoreactive insulin (IRI) were recovered in two peaks, the second common peak representing proinsulin and amounting to 6.5 to 8.4% of total IRI. Immunoreactive glucagon was eluted in a single peak. Chromatography of plasma extracts from two infants of diabetic mothers demonstrated that proinsulin accounted for 59-63% of total IRI, while insulin was separated into two peaks corresponding to the standards of human insulin and porcine insulin. These results indicate that reversed -phase HPLC is a method with a good reproducibility and a high recovery applicable to the rapid and effective separation of pancreatic peptides from biological extracts.     

A rapid high-performance liquid chromatography purification method of lodinated polypeptide hormones

A reverse-phase high-performance liquid chromatography procedure is presented for the separation of a variety of monoiodinated peptides from the corresponding unlabeled and diiodinated hormones. In the case of peptides lacking Met residues, susceptible to oxidation by the chloramine-T-labeling method, chloramine T treatment alone had no effect on elution position of unlabeled hormone. For those hormones containing Met, however, more than 90% of the peptide was transformed into the earlier-eluting oxidized form after 20 to 40 s of chloramine-T treatment. Upon iodination, monoiodinated derivatives were well separated from the corresponding chloramine-T-treated standards, the extent of separation being decreased with increased molecular weight. Application of this technique to the purification of monoiodinated ¹²⁵I-γ-melanocyte-stimulating hormone permitted a threefold increase in titer and sensitivity of a γ-melanocyte-stimulating hormone radioimmunoassay system.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Some applications of reversed-phase high-performance liquid chromatography to oligosaccharide separations

Oligosaccharide separation on reversed-phase high-performance liquid chormatographic columns have been examined using a range of aqueous solvents. Addition of anionic, cationic and non-ionic surfactants, tetramethyl urea and organic solvents to the mobile phase cause faster elution of oligosaccharides, and allow the separation of the larger oligomers in an acceptable time. Addition of neutral, inorganic salts increase the retention factors considerably, and allows good resolution of some compounds poorly resolved in water alone.The mechanism operating in the separations approximates to that invoked in the solvophobic theory of reversed-phase chromatography. There is some evidence also of hydrogen bond effects. The improvements described should prove useful in the isolation and analysis of neutral oligosaccharides in general, and in structural analyses of polysaccharides in paritcular.     

Enantiomeric separation of organophosphorus pesticides by high-performance liquid chromatography,gas chromatography and capillary electrophoresis and their applications to environmental fate and toxicity assays

In recent years, the continuous evolution of the field of stereochemistry has produced a heightened awareness of the applications of pure enantiomers of agrochemicals. This review describes reports of the enantiomeric separation of commercial organophosphorus pesticides (OPs) and the applications of these methods to research on the enantioselectivity of the toxicity and environmental fate of these compounds. Chiral OPs can be analysed by high-performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE). These different separation techniques for OP enantiomers are briefly discussed, and their applications are presented.     

The use of high-performance liquid chromatography for sample clean-up in mass fragmentographic assays

A novel high-performance liquid chromatography (HPLC) sample clean-up procedure for use in mass fragmentographic assays of (sub)-nanogram amounts of drugs in human plasma is described and compared with a conventional extraction sequence for sample purification. With the assay of the new antidepressant drug mianserin hydrochloride (Org GB 94) as an example, the HPLC procedure is discussed with respect to retention time, recovery, purification, column deterioration and convenience. It is demonstrated that HPLC sample clean-up is a useful and time-saving procedure for routine clinical analyses.     

Assay for taurine conjugates of bile acids in serum by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a new high-performance liquid chromatographic (HPLC) procedure for quantifying taurine conjugates of bile acids in serum. The technique involved three basic steps. The first removed free amino acids via solid-phase extraction of the serum. The second step involved the reaction of the extracted serum with the enzyme choloylycine hydrolase, which liberated the taurine from the conjugated bile acids. The third step was the reversed-phase HPLC separation of o-phthalicdicarboxaldehhyde derivatives of taurine. The assay provides a simple technique for determination of the total amount of taurine-conjugated bile acids in serum.     

Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method has been developed for the detection, separation and measurement of codeine and its metabolites norcodeine, morphine and normorphine, with their glucuronide conjugates. The glucuronidase Escherichia coli type VIIA hydrolyses codeine-6-glucuronide completely and is used for the construction of the calibration curves of codeine-6-glucuronide. Enzymic hydrolysis of codeine-6-glucuronide depends on the specific activity of the glucuronidase applied. Examples are shown of a volunteer who is able to form morphine from codeine and one who is unable to do so.     

high-performance liquid chromatography of amino acids, peptides and proteins : LXIII. Reversed-phase high-performance liquid chromatographic characterisation of several polypeptide and protein hormones

The chromatographic behaviour on alkylsilicas of a variety of hormonal proteins is described. Optimization of resolution and recovery of these protein hormones, which included porcine relaxins, human chorionic gonadotropin, human placental lactogen, pituitary derived growth hormone and adenohypophyseal glycoprotein hormones, was achieved by manipulation of both mobile and stationary phase parameters. With standard stainless-steel analytical columns (10–30 cm × 0.4 cm) packed with meso- or macro-porous n-alkylsilica supports these proteins can be readily fractionated at the semi-preparative level with separation times generally under 90 min using elution systems directly compatible with subsequent methods of primary structure determination or biological functional analysis. The effects of changes in several experimental parameters on peak symmetry, retention and recovery are described.     

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.     

Strontium-phosphate hydroxyapatite high-performance liquid chromatography

High-performance liquid chromatography using spherical aggregates of strontium-phosphate hydroxyapatite(SrHA) micro-crystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. It can be deduced that, in parallel with the case of usual calcium-phosphate hydroxyapatite(CaHA), with SrHA also, two types of effective surface, vector a (or vector b) and vector c surfaces, appear on the crystal: the same protein molecular generally shows slightly different chromatographic behaviors between the CaHA and the SrHA packed column. Combining the SrHA and the CaHA packed column would lead to an efficient fractionation of a particular molecule from an assembly of molecules with subtle structural differences from one another.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C₁₈ column with isocratic elution with acetate buffer (25 mM, pH 4.5)—methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35°C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 μg/ml for BA and HA and 5.0 μg/ml for PAA and PA with a 20-μl injection.     

Quantitative analysis of monosialogangliosides by high-performance liquid chromatography of their perbenzoyl derivatives

A quantitative high-performance liquid chromatographic method for the analysis of monosialogangliosides as their perbenzoyl derivatives has been devised. Samples containing as little as 3 nmol were converted to their perbenzoyl derivatives by reaction with 0.1 ml of 10% benzoyl chloride in pyridine at 60 degrees C for 1 hr. The products were purified by silicic acid chromatography and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a 50 cm X 2.1 mm LiChrosphere SI 4000 column and a linear gradient of 7-23% dioxane in hexane in 18 min. Detection was at 230 nm. The detector response was found to be proportional to the amount of monosialoganglioside analyzed. As little as 50 pmol of injected material could be conveniently quantitated. The overall yield from derivatization and chromatography, as determined with radiolabeled GM1, was found to be 86%. To take advantage of the high sensitivity of the HPLC, a small-scale isolation method for gangliosides was devised. This method coupled with HPLC isotope dilution analysis was used to analyze the GM3 content of 1 ml of human plasma.     

Analysis of polyamines as their dabsyl derivatives by reversed-phase high-performance liquid chromatography

The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.     

Analysis of reducing sugars as their chromophoric hydrazones by high-performance liquid chromatography

A chromophoric hydrazide, 4'-N,N-dimethylamino-4-azobenzene sulfonyl hydrazide (DABS-hydrazide), was prepared from 4'-N,N-dimethylamino-4-azobenzene sulfonyl chloride by reaction with hydrazine. Reducing sugars were derivatized with DABS-hydrazide at 50 degrees C for 120 min. The chromophoric hydrazones were separated by reversed-phase HPLC isocratically using a short column (4.6 X 50 mm) and 0.08 M acetic acid-acetone as an eluant with no sample pretreatment and were quantitated at the picomole level. This method was applied to the sugar analysis of 5 micrograms of glycoproteins. Dansyl hydrazide derivatives of sugars were also separated by this HPLC system.