High-performance Liquid Chromatographic Isolation Research Articles

HPLC separation, synthesis, isolation and characterization of process related and degradation impurities in larotaxel including method development and validation

In the synthesis of larotaxel, a new-generation toxoid, eleven related impurities were detected. In this study, Impurity-I, II, III, IV, VII, IX, X and XI were synthesized, and Impurity-VI, VIII were isolated with the help of preparative high-performance liquid chromatography (HPLC). The structures of all impurities were characterized using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectral data, and the possible origins of them were explained. Furthermore, a sensitive and accurate HPLC method was developed for the determination of larotaxel and its eleven impurities. The method was validated to fulfill the requirements of the International Conference on Harmonisation (ICH) guidelines, including specificity, sensitivity, precision, accuracy, linearity, and robustness. The validated method can be applied for routine quality control analysis of larotaxel.

SLE Single-Step Purification and HPLC Isolation Method for Sterols and Triterpenic Dialcohols Analysis from Olive Oil.

The unsaponifiable fraction of oils and fats constitutes a very small fraction but it is an essential part of the healthy properties of some specific oils. It is a complex fraction formed by a large number of minor compounds and it is a source of information to characterize and authenticate the oil sample. Specially, the composition of sterols of any oil or fat is a distinctive feature of itself and, therefore, it has become a useful tool for detecting contaminants and adulterants in oils. A new supported liquid extraction (SLE) technique for the analysis and characterization of the unsaponifiable fraction of fats and oils is proposed. The SLE system includes, as a stationary phase, a combination of adsorbent materials which allow a highly purified unsaponifiable matter ready to be isolated by high performance liquid chromatography (HPLC) and quantified by gas chromatography (GC). This method ensures the removal of fatty acids, avoiding possible interferences and making the analysis of sterols and triterpenic dialcohols easier. The procedure uses a small sample size (0.2 g), reduces the volume of solvents and reagents, and reduces the handling of samples subjected to analytical control. All this is achieved without losing either precision—a relative standard deviation of each compound lower than the reference value (≤16.4%)—or recovery, being for all compounds higher than 88.00%. Therefore, this new technique represents a significant economic and time saving in business control laboratories, a larger productivity and enhancement of working safety.

Evaluation of δ13C and δ15N Uncertainties Associated with the Compound-Specific Isotope Analysis of Geoporphyrins.

Compound-specific isotope analyses of geoporphyrins, which are derivatives of chloropigments possessed by phototrophs, provide essential records of the biogeochemical cycle of past aquatic environments. Here, we evaluated uncertainties in carbon and nitrogen isotopic compositions (δ13C and δ15N) associated with high-performance liquid chromatography (HPLC) purification and isotopic measurements of geoporphyrins. Evaluation of total blank carbon and nitrogen for the HPLC and our sensitivity-improved elemental analyzer/isotope ratio mass spectrometer (nano-EA/IRMS) analysis confirmed that blank carbon can be corrected and that blank nitrogen is negligible compared to the mass of geoporphyrins required for the isotopic measurement. While geoporphyrins exhibited substantial in-peak carbon and nitrogen isotopic fractionations, no systematic changes in δ13C and δ15N values were observed during reversed- and normal-phase HPLC isolation of Ni- and VO-porphyrin standards, with the changes in δ13C and δ15N values being within ±0.6‰ and ±1.2‰ (2σ), respectively. These values are comparable to the instrumental precision of the nano-EA/IRMS system (±1.3‰ for 0.70 μgC and ±1.1‰ for 0.08 μgN, 2σ), confirming that no substantial artifact in the δ13C and δ15N values would be expected during the reversed- and normal-phase HPLC purification. The sensitivity and precision of our method enable us to determine δ13C and δ15N values of both major and minor geoporphyrins found in ancient sediments, which would provide detailed information on the photosynthetic primary producers and the carbon and nitrogen cycles in the past.

English

The aerial parts of Capsella bursa-pastoris (L.) Medic are used to treat nephritis, edema and enteritis. Every barber knows that C. bursa-pastoris had a good anti-inflammatory effect. This suggests that the extracted components from C. bursa-pastoris could potentially treat inflammatory disease. For discovering of the anti-inflammatory effects and chemical composition of C. bursa-pastoris, EtOAc extract was extracted from C. bursa-pastoris (EECB) and researched on EECB’s anti-inflammatory effects. On the carrageenan-induced paw oedema experiment, the EECB used at the doses (100, 200 and 300 mg/kg) after 10 h (p < 0.01), 5 h (p < 0.01) and 3 h (p < 0.01), respectively, showed significant anti-inflammatory effects. Moreover, on the egg-albumin-induced inflammation experiment, the EECB used at the doses 200 and 300 mg/kg after 4 h (p < 0.01) and 2 h (p < 0.01), respectively, showed significant anti-inflammatory effects. In accordance with the HPLC isolation of the EECB, there are four major compounds, namely, apigenin-7-O-β-D-glucopyranoside (S1), luteolin-7-O-β-D-glucopyranoside (S2), α-adenosine (S3), and uridine (S4), which may explain the activity. Key words: Capsella bursa-pastoris (L.) Medic, anti-inflammatory, high performance liquid chromatography (HPLC) isolation, flavonoids.

Carbon Stable Isotope Analysis for Glucose in Sake: Simple Freeze-Dried Sake Can Substitute for Glucose Following HPLC Isolation

Carbon stable isotope analysis of ethanol is an established method for determining the authenticity of alcoholic beverages, but there is no accepted method for measurements of glucose carbon stable isotopic composition (δ 13C). This study aims to establish two methods to determine accurately the δ 13C value of glucose isolated from sake by high-performance liquid chromatography (HPLC) after three clean-up steps using ion exchange chromatography and in simple freeze-dried sake (total nonvolatile matter) and to examine two methods that yield different δ 13C values. In the glucose isolation procedure, the δ 13C values of glucose were not significantly affected by the glucose concentration, solvent (water or aqueous acetonitrile), drying method (freeze-dry or nitrogen-spraying), the clean-up step using ion exchange chromatography (weakly acidic cation-exchange, strongly acidic cation-exchange, and weakly basic anion-exchange resins), or HPLC isolation. Glucose nitrogen spray-dried after isolation by ion exchange chromatography and HPLC showed a carbon isotope discrimination value of <0.1 ‰. The glucose δ 13C values ranged from −26.8 to −25.5 ‰ in authentic sake and from −27.0 to −11.0 ‰ in commercial sake samples. This HPLC isolation approach may provide a first step toward visualizing the brewing process via isotopic carbon flow during fermentation. Additionally, there was no significant difference between the δ 13C values of glucose and total nonvolatile matter from sake. The δ 13C measurement of simple freeze-dried sake can substitute for glucose δ 13C measurement using HPLC isolation, thus aiding high-throughput detection of carbon derived from C4 plants in sake.

Determination of methyl gallate in Bauhinia retusa extract by high-performance liquid and thin-layer chromatography

ABSTRACTTwo stability-indicating chromatographic methods are reported for the determination of methyl gallate in crude extracts of Bauhinia retusa. Separation by high performance thin layer chromatography was conducted on silica gel aluminum sheets using 9.5:0.5:0.2 (v/v/v) chloroform:methanol:acetic acid at 280 nm. The results from the 2–40 µg/band were used to prepare a linear calibration graph. The limits of detection and quantitation were 0.5 and 1.5 µg/band, respectively. The reverse phase high performance liquid chromatographic isolation of methyl gallate was performed at ambient temperature with an injection volume of 10 μL. The mobile phase consisted of 40:60 (v/v) methanol:0.1% ortho-phosphoric acid. The separation was performed at 1 mL/min using a detection wavelength of 280 nm. The calibration graph for methyl gallate was rectilinear from 0.02–40 µg/mL with limits of detection and quantitation of 0.004 and 0.010 µg/mL, respectively. For both methods, intra-day and inter-day precision were evaluated and the relative standard deviation was less than 2%, indicating good precision. The robustness was evaluated by making small and deliberate changes to appropriate parameters and the calculated relative standard deviation was less than 2%.The chromatographic methods were employed to determine methyl gallate in crude Bauhinia retusa extracts.

Invariant chlorine isotopic signatures during microbial PCB reductive dechlorination

In order to develop more robust insight into the natural attenuation of polychlorinated biphenyls (PCBs), the chlorine isotopic composition of residual 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) was monitored as it underwent microbial reductive dechlorination to 2,3,5-trichlorobiphenyl (2,3,5-CB) in laboratory cultures. Reverse-phase high performance liquid chromatography (HPLC) was employed to isolate the former compound from the experimental matrix for δ 37Cl measurement. No detectable isotopic fractionation was observed over the 90 day incubation with sterile control, standard, and inoculated samples all exhibiting δ 37Cl values with a range of ∼0.5‰. These results show that this type of biological activity can be discriminated from other transformations by the absence of a measurable isotope effect during microbial reductive dechlorination. The utility of HPLC isolation for compound-specific δ 37Cl analyses of environmentally relevant species is also demonstrated.

Serotonin (5-hydroxytryptamine) glucuronidation in vitro : assay development, human liver microsome activities and species differences

1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to β-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5- O -glucuronide by 1 H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K m and V max of 8.8 ±0.3 mM and 43.4 ±0.4 nmoles min <1 mg <1 protein, respectively, for human liver microsomes, and 5.9 ±0.2 mM and 15.8 ±0.2 nmoles min <1 mg <1, respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro.

Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein.

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.

The use of heptafluorobutyric acid as a volatile ion-pair reagent in the high-performance liquid chromatographic isolation of SB-223070

The separation of the E-oxime (SB-223070) from the Z-oxime (SB-220582) was achieved using the ion pairing reagent sodium dodecyl sulfonate. When a pure sample of SB-223070 was required by preparative chromatography from mother liquors, modification of the chromatographic conditions was necessary. The capacity factor and resolution between the two oximes needed to be increased to isolate the E-oxime from the other components in the mother liquor and a method to remove the ion-pairing reagent from the isolated product was required. The volatile ion pairing reagent trifluoroacetic acid did not retain or resolve the components sufficiently but by switching to heptafluorobutyric acid, isolation of SB-223070 was achieved as the heptafluorobutyrate. This salt was liberated by hydrochloric acid to give the required hydrochloride salt and the remaining heptafluorobutyric acid was removed by extraction into dichloromethane.

High Performance Liquid Chromatographic Isolation and Spectroscopic Characterization of Metabolites from the Bile of Rats Receiving Rapamycin (Sirolimus) Intravenously

Ten major metabolites of rapamycin (M2, M3, M8, M9, M10, M11, M13, M14, M15, and M16) were isolated from pooled bile of intravenously dosed rats. Metabolites were extracted from the bile with ethyl acetate prior to isolation by HPLC using a Supelcosil SPLC-18, μm, 10 × 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS analyses. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M10 is oxygenated in the southern portion of rapamycin and the macrolide ring is intact. M13 is a monohydroxylation and demethylation metabolite and both biotransformations occurred at the southern portion. M8, M9, and M11 are monohydroxylation and demethylation metabolites. M14 and M15 are di-hydroxylation metabolites. M16 is mainly a dihydroxylation metabolite. † This work was presented at the Fourth International ISSX Meeting, Seattle, WA, August 1995.

High Performance Liquid Chromatographic Isolation and Spectroscopic Characterization of Three Major Metabolites from the Plasma of Rats Receiving Rapamycin (Sirolimus) Orally

Abstract Three major metabolites of rapamycin (M2, M3, and M5) were isolated from pooled plasma of orally dosed rats. Metabolites were extracted from the plasma with ethyl acetate/methanol prior to isolation by HPLC using a Supelcosil SPLC-18, 5μm, 10 × 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M5 is O-demethylated on the C41 methoxy moiety and the macrolide ring is intact.

High Performance Liquid Chromatographic Isolation, Spectroscopic Characterization, and Immunosuppressive Activities of Two Rapamycin Degradation Products

Abstract A high performance liquid chromatographic method has been developed for the isolation of two degradation products of rapamycin which is currently under development as an immunosuppressive agent. Prior to isolation, the drug was incubated at 37°C in rat bile or ammonium acetate (pH 8.0). The isolation was achieved by a Supelco, PLC-18 21.2 × 250 mm, 18 μm column using methanol/ammonium acetate gradient mobile phase. After evaporation of methanol, the remaining eluates were lyophilized. The isolated degradation products were characterized by negative ion fast atom bombardment mass spectrometry (FAB MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). Degradation product A was found to be a macrolide ring-opened hydrolysis product of rapamycin where the C25 ester bond had been hydrolyzed. Degradation product B was determined to be a ring-opened isomer of rapamycin. B had less than 4% of the potency of rapamycin in a thymocyte proliferation assay, while A had minimal activity at concentra...

Multidimensional evaluation of impurity profiles for generic cephalexin and cefaclor antibiotics

A multidimensional approach is described for characterizing impurities in samples of generic cefaclor and cephalexin antibiotics. High-performance liquid chromatography (HPLC) with gradient elution followed by photodiode array or mass spectrometric detection provides valuable information concerning the nature of impurities observed. Results are presented which demonstrate the utility of these techniques for identifying impurities and distinguishing among process-related impurities, degradation products and formulation excipients. Preparative HPLC isolation and spectroscopic identification of some impurities is also described.

Ion-exchange high-performance liquid chromatographic isolation of the major allergen of parietaria pollen extract

The analysis and purification of a parietaria pollen extract were attempted using an ion-exchange high-performance liquid chromatographic method on a preparative scale. A homopolymerization equilibrium was observed to occur in solution. A combination of electrophoretic methods and immunoelectrophoretic methods was also used and showed that a small amount of the major allergenic component was isolated.

Positive and negative ion252Cf plasma desorption mass spectrometry of polar agrochemical metabolites

The use of 252Cf plasma desorption (PD) mass spectrometry for the identification of highly polar agrochemical metabolites is illustrated with metabolites of azinphos-methyl, -ethyl, demeton-S-methyl and demeton-S-methyl sulphoxide. Three different classes of metabolites were used, namely the monosodium salt of organophosphorus acid ester, the glycoside and the sodium salt of sulphonic acid. Positive and negative ion mass spectra were recorded for different sample preparation techniques. The electrospray method and nitrocellulose adsorption with sample application by spin-drying with and without a rinsing step was applied and evaluated. PD mass spectrometry has proved to be useful in providing fast information on the molecular weight of all three classes of metabolites, but the method has failed to give fragment ions. The best result in terms of sensitivity and low background were obtained using the nitrocellulose spin-drying method without a washing step. It has been found to be a very valuable technique for the rapid identification of non-volatile or thermally labile agrochemical metabolites after high-performance liquid chromatographic isolation from plants.

Strategy for the preparative-scale high-performance liquid chromatographic isolation of kadsurenone and futoquinol from the medicinal plant Piper futokadsura

The chemical constituents of a Chinese herbal plant, haifenteng ( Piper futokadsura), have been investigated. In addition to terpenes and lignans of diverse chemical structure, a potent receptor antagonist of platelet activating factor (PAF), kadsurenone, has been isolated and identified. PAF is a highly potent lipid mediator of acute inflammation, allergy and anaphylaxis. Its receptor antagonists are potential therapeutic agents for asthma and various cardiovascular and inflammatory disorders. More recently, a plant sample from Taiwan was re-examined in detail by normal-phase open column and high-performance liquid chromatography. The co-elution of sigificant amounts of futoquinol, a known lignan previously isolated from P. futokadsura, hampered the isolation of kadsurenone. To facilitate the isolation of kadsurenone a separation of futoquinol and kadsurenone from a methylene chloride extract of plant was developed on 10-μm silica using gradient chromatography with hexane—methylene chloride—ethyl acetate—acetonitrile. Silica packings of 10, 15 and 37–55 μm were evaluated with this gradient and the 15-μm silica was selected for the initial fractionation of plant extract. The kadsurenone-containing fractions from the normal-phase separation were re-chromatographed using reversed-phase chromatography. A 15-μm C 18 column using water—methanol and water—acetonitrile mobile phases yielded pure kadsurenone.

Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta.

Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.

Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain

A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine (3-MT), noradrenaline, 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40% wt/wt) and graphite powder (60% wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24-67% (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20-60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8-16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed-phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G-10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HClO4, and the excess of HClO4 was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the urinary cortisol production rate using (1,2,3,4-13C)cortisol. Isotope dilution analyses at very small enrichments.

An isotope dilution mass spectrometric method to determine the urinary cortisol production rate (CPR) in babies and children is described. The method uses stable isotopically labelled (1,2,3,4-13C)cortisol. The tracer is intravenously administered to the patient and urine is collected for the following three days. Following extraction, enzymic hydrolysis, purification and isolation by high-performance liquid chromatography (HPLC) the urinary cortisol metabolites tetrahydrocortisone, tetrahydrocortisol, alpha- and beta-cortolone are separately oxidized the common product, 11-oxo-aetiocholanolone. The methyl oxime tert-butyldimethylsilyl ether derivative (MO TBDMS) was analysed by gas chromatography mass spectrometry. Quantification of the isotope enrichment was carried out by unlabelled, and at m/z 348 for labelled 11-oxo-aetiocholanolone. HPLC isolation of the metabolites together with the oxidation step allowed very small isotope enrichments, sometimes down to 0.1% (1:1000), to be reliably measured against a linear calibration graph containing 0 to 1% (13C4) enrichments. The standards for the calibration graph were synthesized from mixtures of labelled (13C4) cortisol and natural cortisol, and the calibration graph was prepared each time samples were measured. The long term instrumental precision of the isotope dilution analyses was 0.91% for a derivatized sample containing a (13C4) enrichment of 0.5% (measured on six different days over seven months). The coefficient of variation of the complete procedure for the four cortisol metabolites was between 1.17 and 2.14%. The clinical applicability of the method is demonstrated by presenting the results of a CPR determination in a patient.