Reversed-phase high-pressure liquid chromatography of arachidonic acid metabolites formed by cyclooxygenase and lipoxygenases

High-pressure liquid chromatography is required to resolve the complex mixtures of arachidonic acid metabolites synthesized by many tissues. We have investigated some of the factors which affect the retention times of these substances in reversed-phase HPLC on columns of 5-micron octadecylsilyl silica. There are considerable differences in selectivity between mobile phases based on methanol and those based on acetonitrile, the latter being much better for cyclooxygenase products. The chromatographic behavior of peptidoleukotrienes (LTC4, LTD4, and LTE4) is quite different from that of other arachidonic acid metabolites which do not contain amino acids. Addition of phosphoric acid to the mobile phase results in very long retention times for peptidoleukotrienes. Very low concentrations of trifluoroacetic acid have effects similar to that of phosphoric acid, but as its concentration is raised, the retention times of peptidoleukotrienes decrease, whereas those of other arachidonic acid metabolites are unaffected. Changing the concentration of acetonitrile in the mobile phase also affects the retention times of peptidoleukotrienes differently from those of other metabolites. This information has been used to devise simple linear gradients which separate most of the major cyclooxygenase and lipoxygenase products of arachidonic acid metabolism.     

Identification of the major vanilloid component in Capsicum extract by HPLC-EC and HPLC-MS

A sensitive multi-channel HPLC-electrochemical (EC) method has been developed to determine the vanilloid content in the complex Capsicum annuum extract Capsibiol. Chromatographic separation was achieved within 10 min using a YMC Basic S5 column with a mobile phase containing chloroacetic acid, heptane sulphonic acid and acetonitrile. The multi-channel detector simultaneously applied four potentials between +500 and +800 mV (referenced to a silver/silver chloride electrode) to four glassy carbon working electrodes. The most abundant (0.94 mg/g) vanilloid analogue in the Capsibiol sample demonstrated an electrochemical reactivity and retention time similar to that of vanillic acid in HPLC-EC analysis. Its identity was confirmed by HPLC-MS using a Zorbax SB-CN column with a mobile phase containing formic acid and methanol.     

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.     

A simple, sensitive method for measuring 3,4-dihydroxyphenylacetic acid and homovanillic acid in rat brain tissue using high-performance liquid chromatography with electrochemical detection.

A sensitive, specific, and very simple method, using high-performance liquid chromatography combined with electrochemical detection, measured 3,4-dihydroxyphenylacetic acid and homovanillic acid in small areas of rat brain. The dopamine metabolites were extracted with diethyl ether and a known amount of vanillic acid as internal standard, and were separated on a micro-particulate reverse-phase column using a sodium acetate buffer (pH 5.0) as mobile phase.     

Determination of a prostaglandin D2 antagonist and its acyl glucuronide metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometric detection--a lack of MS/MS selectivity between a glucuronide conjugate and a phase I metabolite

A method for the determination of a prostaglandin D(2) receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k') of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k' increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4 degrees C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10-2500 ng/mL for both drug and glucuronide metabolite.     

Liquid chromatographic determination of free and conjugated 3-methoxy-4-hydroxyphenylethylene glycol with wide-ranging substances related to monoamine metabolism

A simple and sensitive procedure was developed for the simultaneous determination of substances metabolically related to monoamine transmitters including 3-methoxy-4-hydroxy-phenylethylene glycol (MOPEG) in dissected brain regions of rats using high-performance liquid chromatography combined with electrochemical detection. The tissue sample was homogenized in HCl solution. The homogenate was divided into two portions, of which one was used for the assay of MOPEG after enzymatic hydrolysis with sulfatase. A butanol extraction process was performed on the remaining portion to obtain the sample of monoamine transmitters, precursor amino acids, and acidic metabolites. The monoamines and precursor amino acids were finally recovered in HCl solution, while the acidic metabolites shifted into the alkaline buffer from the organic layer. The basic and neutral substances were separated with a 0.1 M sodium citrate/citric acid buffer system (pH 4.0) containing 1% tetrahydrofuran, and the acidic ones with 0.075 M sodium citrate/citric acid buffer (pH 3.5) containing 1% tetrahydrofuran, 10% methanol, and 12% acetic acid. The steady-state concentrations of three monoamine transmitters (noradrenaline, dopamine, and 5-hydroxytryptamine) were determined together with their precursors and metabolites. Changes in the concentrations of these substances were examined for various drugs, of which the effects had been previously confirmed. The changes reflected putative drug effects and demonstrated that the procedure was applicable to the regional determination of monoamines and their metabolically related substances.     

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The α-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 μM vanilmandelic acid.     

Simple and rapid determination of serotonin and catecholamines in biological tissue using high-performance liquid chromatography with electrochemical detection

Using the CNS of Lymnaea stagnalis a method is described for the rapid analysis of neurotransmitters and their metabolites using high performance liquid chromatography coupled with electrochemical detection. Tissue samples were homogenised in ice-cold 0.1 M perchloric acid and centrifuged. Using a C(18) microbore column the mobile phase was maintained at a flow rate of 100 microl/min and consisted of sodium citrate buffer (pH 3.2)-acetonitrile (82.5:17.5, v/v) with 2 mM decane-sulfonic acid sodium salt. The potential was set at +750 mV versus Ag|AgCl reference electrode at a sensitivity of 50 nA full scale deflection. The detection limit for serotonin was 11.86 ng ml(-1) for a 5 microl injection. Preparation of tissue samples in mobile phase reduced the response to dopamine and serotonin compared with perchloric acid. In addition it was found that the storage of tissue samples at -20 degrees C caused losses of dopamine and serotonin. As a result of optimising the sample preparation and mobile phase the total time of analysis was substantially reduced resulting in a sample preparation and assay time of 15-20 min.     

Method for the analysis of the methylphosphonic acid metabolites of sarin and its ethanol-substituted analogue in urine as applied to the victims of the Tokyo sarin disaster

An analysis method for the methylphosphonic acid metabolites of sarin in urine using trimethylsilyl derivatization and flame photometric detection is described in this report. Authentic reference standards of isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) as well as methylphosphonic acid were employed to estimate the concentration in human urine. A sample pretreatment procedure was developed for urine using a column of cation-loaded ion-exchange resins (Ag⁺-, Ba²⁺- or H⁺-Dowex) and adjusting the pH of the eluate from the column to 3.75–3.85 improved recovery of the target compounds. The eluate was evaporated to dryness under vacuum prior to trimethylsilylation, to remove water and any hydroxy- or amino-carrying volatile substances. The sarin metabolites, because of their low volatility, were concentrated and could be derivatized for analysis. The use of synthesized authentic sarin and ethylsarin metabolites, i.e., IMPA and EMPA, made it possible to establish the necessary sample pretreatment procedures for derivatization and gas chromatography–flame photometric detection (GC–FPD) analysis. The detection limits were 0.025 ppm both for EMPA and IMPA, and 0.625 μM for MPA, respectively. This method can be useful for estimating the exposure level to sarin by assaying the metabolites in urine and it is applicable to a large numbers of samples.     

Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH

Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Determination of dopamine, homovanillic acid and 3,4-dihydroxyphenylacetic acid in rat brain striatum by high-performance liquid chromatography with electrochemical detection

Two procedures using liquid chromatography with electrochemical detection are described for the determination of dopamine (DA) and its two acidic metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), in subregions of rat striatum and nucleus accumbens. A strong cation-exchange column was used for DA analysis and a C₁ reversed-phase column was used for the analysis of the metabolites. Effects of pH, temperature and percentage of methanol on the retention time of HVA and DOPAC were studied. Levels of these compounds in the subregions of rat striatum and nucleus accumbens are reported.     

Simultaneous measurement of dopamine and its metabolites, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in brain tissue using liquid chromatography and electrochemical detection

An assay has been developed for brain tryptophan using reverse-phase liquid chromatography with electrochemical detection. The method simultaneously assays dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). The method does not require elution from ion exchange resins. After deproteinization and centrifugation samples are injected directly onto the chromatographic column. It was found that small changes in mobile phase pH markedly influenced the retention time of tryptophan while elution of the indoleamines and catecholamines did not change. The assay of these endogenous compounds in a single injection proved not expedient but inexpensive. Values obtained using alumina and ion exchange resins yielded comparable values.     

Porous graphitic carbon shows promise for the rapid chromatographic analysis of polar drug metabolites

A rapid and simple HPLC method has been developed and used to separate the polar metabolic conjugates of AZT, chloramphenicol, and β-estradiol based upon the use of porous graphitic carbon. The HPLC system is sufficiently selective to resolve the polar drug conjugates from their parent compounds and from endogenous material present in urine. The compounds are separated, without the need for sample pretreatment or gradient elution, on a porous graphitic carbon (Hypercarb) column using aqueous trifluoroacetic acid modified with tetrahydrofuran as the mobile phase. Porous graphitic carbon exhibits a novel mechanism of retention towards these very polar substances, which are unretained under reversed-phase conditions on alkyl-bonded silica phases.     

Analysis of acetaminophen metabolites in urine by high-performance liquid chromatography with UV and amperometric detection

Acetaminophen and several of its metabolites are separated isocratically on a reversed-phase (C₁) column using a mobile phase of 7% methanol and 0.75% glacial acetic acid in 0.1 M KH₂PO₄. Metabolites that can be separated include the sulfate, glucuronide, cysteine, and mercapturic acid conjugates of acetaminophen, as well as 3-hydroxyacetaminophen, 3-methoxyacetaminophen, and 3-methylthioacetaminophen. Although all of the metabolites can be detected by UV spectrophotometry, the sensitivity limits of detection are improved significantly for acetaminophen and all of the metabolites except the sulfate and glucuronide, by amperometric detection (electrochemical) of the same sample as it elutes from the UV detector. Minimal detectable limits (signal-to-noise ratio 2) for acetaminophen and its metabolites, other than the glucuronide and sulfate conjugates, were in the order of 1–2 ng injected on column using UV detection at 248 nm, and 0.1–0.5 ng using electrochemical detection at + 0.60 V with reference to an Ag/AgCl standard electrode.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.     

In-use stability of monoamine metabolites in human cerebrospinal fluid

Cerebrospinal fluid (CSF) concentrations of the monoamine metabolites homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) are commonly used to provide information about central nervous system (CNS) dopaminergic and serotonergic activity. However, little attention has been given to the effects of sample handling on the concentrations of these compounds in human CSF. Using high-performance liquid chromatography (HPLC) with electrochemical detection, we observed that, in CSF stored at −80°C, concentrations of the serotonin metabolite 5-HIAA and the dopamine metabolite HVA remained unchanged through six 1-h and six 24-h freeze–thaw cycles. Exposure to bright room light (3 h, 1230 lux) resulted in a 5-HIAA concentration that was 96.3±2.0% of the initial and an HVA concentration that was 98.8±1.03% of initial. The pH of the CSF significantly increased during both freeze–thaw series and while maintained on ice (4°C). These results demonstrate the in-use stability of 5-HIAA and HVA in human CSF under commonly-encountered laboratory conditions.     

Determination of cocaine, benzoylecgonine and ecgonine methyl ester in plasma by reversed-phase high-performance liquid chromatography

A combined assay is described for cocaine and its major metabolites, benzoylecgonine and ecgonine methyl ester. The method uses electrochemical and ultraviolet detectors in series. A non-silica column is used with high-pH mobile phase. The three compounds are completely separated from other cocaine metabolites. The assay has been suitable for pharmacokinetic studies of cocaine disposition in animal studies.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 1000 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients on variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at −25°C prior to analysis.