Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection

A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70-80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly 3H2O. The eicosanoid metabolites were fractionated into different polarity classes using reverse phase Sep-Pak C18 cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime-tert-butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotopic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

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.     

Simultaneous detection of methylphenidate and its main metabolite, ritalinic acid, in doping control

Two analytical methods for the simultaneous detection in urine of methylphenidate and its main metabolite, ritalinic acid, are described. Both procedures are based on solid-phase extraction of urine samples on Bond Elut Certify columns, and capillary gas chromatographic—mass spectrometric detection of O-trimethylsilyl, N-trifluoroacetyl derivatives. The former method is used as a general screening procedure for the detection of basic polar nitrogen-containing compounds in urine such as stimulants, narcotic and adrenergic drugs. The latter procedure is proposed as a specific method to confirm methylphenidate ingestion. The two methods are sensitive enough to detect methylphenidate and ritalinic acid in urine at least for 24 h after administration of a therapeutic dose (20 mg oral dose) of methylphenidate.     

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.     

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.     

A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system.

A rapid and sensitive method for separation and concurrent assay of 14 compounds at the picomole level in individual rat brain parts is described. The putative amino acid neurotransmitters (aspartate, γ-aminobutyric acid, glutamate, and glycine) are extracted from a 20–30 mg portion of the tissue with 5% TCA and assayed as their respective DNP-amino acid methyl ester derivatives by glc. Four other putative neurotransmitters (acetylcholine, dopamine, norepinephrine, and serotonin) and some of their precursors and metabolites (choline, tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and tyrosine) are extracted in 1n formic acid-acetone (v/v:15/85) from the remaining tissue. The lipids are removed with a heptane-chloroform (v/v:8/1) wash and the aqueous phase is dried at 37°C under N₂. The dried extract is dissolved in water (pH 4). With one portion of this solution, acetylcholine and choline are assayed using a radioenzymatic method whereas with the rest, dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, tryptophan, and tyrosine are separated with three ion exchange resins arranged in tandem. These compounds are assayed fluorometrically with modified microadaptations of previously reported methods.     

Chiral resolution of the epoxyeicosatrienoic acids, arachidonic acid epoxygenase metabolites

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.     

Quantitation of a new potent angiotensin II receptor antagonist,TCV-116, and its metabolites in human serum and urine

A sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of a new potent antihypertensive agent, TCV-116, and its two metabolites (M-I and M-II) in human serum or urine. After pre-treatment of the specimens, the analytes were determined using a column switching technique, except for the metabolites in urine which were determined by gradient elution mode HPLC. The quantitation limits for TCV-116, M-I and M-II were all 0.5 ng/ml in serum, and 0.5, 10 and 10 ng/ml in urine, respectively. The methods were applied to clinical trials of TCV-116.     

Determination of uric acid by an anion-exchange column chromatography

A column chromatography using a conventional anion-exchange resin for the separation of uric acid from other purine metabolites is described. It uses a HCl gradient, and the amount of uric acid is quantified directly by monitoring the absorbance of the effluent at 285 nm. The linear range of response is 0.5 to 100 nmol. The method was applied to the analysis of uric acid in urine and serum. Urine was injected directly into the system, while serum required removal of an interfering substance which absorbs the light and coelutes with uric acid. However, this substance was simply removed by heat coagulation of serum by heating in a boiling water bath for 2 min.     

Novel liquid chromatography-electrospray ionization mass spectrometry method for the quantification in human urine of microbial aromatic acid metabolites derived from dietary polyphenols

An HPLC-ESI-MS-MS method was developed to quantify in human urine fourteen aromatic acids known as metabolites of dietary polyphenols. These metabolites were determined simultaneously in a single 20-min chromatographic analysis with multiple reaction monitoring detection. The inter- and intra-day precisions, calculated from quality control samples were 8.8 and 5.3%, respectively, and the mean accuracy was 2.3%. The method was tested on urine samples collected from one healthy volunteer who consumed a polyphenol-rich diet for 3 days. Increased levels of several aromatic acid metabolites were observed, demonstrating that the method can be used to detect changes in the excretion of microbial metabolites induced by the consumption of polyphenol-containing foods in humans.     

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.     

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias <15%, except at the lowest limit of quantification (<20%). The inter-assay coefficients of variation and bias were <15%. The application of this method to plasma and urine samples of five CYP2D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.     

Effect of hippuric acid on the gaschromatographic retention of S-phenylmercapturic acid

S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.     

Metabolic studies on retinoic acid in the rat

The nature of metabolites in the urine arising from differentially labelled retinoic acid was investigated after injection of physiological doses into retinol-deficient rats. Distribution of radioactivity after partition of urine into ether-soluble, acidic and water-soluble fractions revealed that there were at least six metabolites in urine. Of these, the major metabolite(s) was one lacking both C-14 and C-15 of retinoic acid. Enzymic or alkaline hydrolysis of acidic and water-soluble fractions did not release any retinoic acid, thus indicating that retinoyl beta-glucuronide was not present in urine in significant amounts.     

Orally administered rosmarinic acid is present as the conjugated and/or methylated forms in plasma, and is degraded and metabolized to conjugated forms of caffeic acid, ferulic acid and m-coumaric acid

Rosmarinic acid (RA) is contained in various Lamiaceae herbs used commonly as culinary herbs. Although RA has various potent physiological actions, little is known on its bioavailability. We therefore investigated the absorption and metabolism of orally administered RA in rats. After being deprived of food for 12 h, RA (50 mg/kg body weight) or deionized water was administered orally to rats. Blood samples were collected from a cannula inserted in the femoral artery before and at designated time intervals after administration of RA. Urine excreted within 0 to 8 h and 8 to 18 h post-administration was also collected. RA and its related metabolites in plasma and urine were measured by LC-MS after treatment with sulfatase and/or beta-glucuronidase. RA, mono-methylated RA (methyl-RA) and m-coumaric acid (COA) were detected in plasma, with peak concentrations being reached at 0.5, 1 and 8 h after RA administration, respectively. RA, methyl-RA, caffeic acid (CAA), ferulic acid (FA) and COA were detected in urine after RA administration. These components in plasma and urine were present predominantly as conjugated forms such as glucuronide or sulfate. The percentage of the original oral dose of RA excreted in the urine within 18 h of administration as free and conjugated forms was 0.44 +/- 0.21% for RA, 1.60 +/- 0.74% for methyl-RA, 1.06 +/- 0.35% for CAA, 1.70 +/- 0.45% for FA and 0.67 +/- 0.29% for COA. Approximately 83% of the total amount of these metabolites was excreted in the period 8 to 18 h after RA administration. These results suggest that RA was absorbed and metabolized as conjugated and/or methylated forms, and that the majority of RA absorbed was degraded into conjugated and/or methylated forms of CAA, FA and COA before being excreted gradually in the urine.     

A method for the estimation of 6-oxygenated metabolites of progesterone in urine

1. A method is described for the estimation of the 6-oxygenated metabolites of progesterone in urine. After hydrolysis the extract of urine is chromatographed on alumina to obtain a fraction containing mainly the 6-oxygenated metabolites. This fraction is oxidized to convert the metabolites into pregnane-3,6,20-triones, which are estimated as the dinitrophenylhydrazones. 2. The reliability criteria of the method are presented. Normal subjects excrete 0.1-0.6mg./day, and at the end of pregnancy values of 3.3-11.6mg./day are obtained.     

Quantitative determination of histamine metabolites by capillary gas chromatography

A method using capillary gas chromatography is described for the determination of histamine and eight of its basic and acid metabolites in a single biological sample of serum, urine, or gastric juice. Ion-exchange chromatography and extraction with organic solvents are used for isolation and purification, and gas chromatography for identification and quantitation. The heptafluorobutyryl derivatives of histamine and some basic metabolites are compatible with nitrogen-phosphorus and electron capture detection modes and offer an excellent sensitivity (detection limit 0.1 pmol with electron capture). The acid metabolites are quantitated after esterification. The linearity range, the sensitivity, a partial study of reproducibility and typical chromatograms show that the method is adaptable to a variety of applications.