Ion-pair Liquid Research Articles

The Effect of Glutathione Modulation on the Concentration of Homocysteine in Plasma of Rats

Elevated plasma homocysteine concentration in humans is associated with increased risk of arteriosclerosis and ischaemic heart disease. We studied whether the plasma homocysteine concentration could be changed by administration of drugs that modulate the concentration of glutathione in both plasma and tissue. Male wistar rats received reduced glutathione (0.5 mmol/kg), N-acetylcysteine (0.5 mmol/kg), L-buthionine-[S,R]-sulfoximine (2 mmol/kg) or Ringer acetate intravenously. Twenty min. later an arterial blood sample was drawn for the measurement of homocysteine and other thiols in the plasma. The thiols were quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The total homocysteine concentration in plasma of fasted rats was 6.1±0.5 μM. Intravenous administration of reduced glutathione or N-acetylcysteine reduced the homocysteine concentration in plasma significantly by 51% to 3.0±0.3 μM and 63% to 2.2±0.2 μM, respectively (P<0.05). In contrast, L-buthionine-[S,R]-sulfoximine increased the concentration of homocysteine by 41% to 8.6±0.6 μM (P<0.05). The glutathione concentration in plasma was 19.5±1.9 μM in controls and was unchanged by N-acetylcysteine administration. Reduced glutathione increased plasma glutathione to 379.7±22.9 μM (P<0.05), whereas L-buthionine-[S R]-sulfoximine lowered the plasma glutathione concentration to 5.3±0.4 μM. Homocysteine was negatively correlated to the glutathione (r=−0.399, P<0.01) and the cysteine (r=−0.52, P<0.01) concentrations in plasma. Our conclusion is that modulation of the glutathione levels influences the concentration of homocysteine in plasma of rats.

Application of liquid–liquid–liquid microextraction and ion-pair liquid chromatography coupled with photodiode array detection for the determination of chlorophenols in water

A method termed as liquid–liquid–liquid microextraction was utilized to extract chlorophenols from water. The extracted chlorophenols, present in anionic form, were then separated, identified, and quantitated by ion-pair high-performance liquid chromatography with photodiode array detection (HPLC/DAD). For trace chlorophenol determination using HPLC/DAD, the chlorophenolate anion provides a better ultraviolet spectrum for quantitative and qualitative analyses than does uncharged chlorophenol. This is due to the auxochromic effect of the phenolate anion. In the study, experimental conditions such as organic phase identity, acceptor phase volume, sample agitation, extraction time, acceptor phase NaOH concentration, donor phase HCl concentration, salt addition, and UV absorption wavelength were optimized. Relative standard deviations (RSD, 2.3–5.4%), coefficients of determination ( r 2 0.9994–0.9999), and detection limits (0.049–0.081 ng mL −1) of the proposed method were investigated under the selected conditions. The method was successfully applied to analyses of reservoir and tap water samples, and the relative recoveries of chlorophenols from the spiked reservoir and tap water samples were 94.1–100.4% and 87.8–101.2%, respectively. The proposed method is capable of identifying and quantitating each analyte to 0.5 ng mL −1, confirming the HPLC/DAD technique to be quite robust for monitoring trace levels of chlorophenols in water samples.

Analysis of Underivatized Amino Acids in Geological Samples Using Ion-Pairing Liquid Chromatography and Electrospray Tandem Mass Spectrometry

The capability of detecting biomarkers, such as amino acids, in chemically complex field samples is essential to establishing the knowledge required to search for chemical signatures of life in future planetary explorations. However, due to the complexities of in situ investigations, it is important to establish a new analytical scheme that utilizes a minimal amount of sample preparation. This paper reports the feasibility of a novel and sensitive technique, which has been established to quantitate amino acids in terrestrial crust samples directly without derivatization using volatile ion-pairing liquid chromatography and tandem mass spectrometry equipped with an electrospray ionization source. Adequate separation of 20 underivatized amino acids was achieved on a C(18) capillary column within 26 min with nonafluoropentanoic acid (NFPA) as ion-pairing reagent. Each amino acid was identified from its retention time as well as from its characteristic parent-to-daughter ion transition. Using tandem mass spectrometry as a detection technique allows co-elution of some amino acids, as it is more specific than traditional spectrophotometric methods. In the present study, terrestrial samples collected from 3 different locations were analyzed for their water-extractable free amino acid contents, following the removal of metal and organic interferences via ion exchange procedures. This is the first time that amino acids in geological samples were directly determined quantitatively without complicated derivatization steps. Depending on the amino acid, the detection limits varied from 0.02 to 5.7 pmol with the use of a 1 microl sample injection loop.

Ultraperformance Ion-Pair Liquid Chromatography Coupled to Electrospray Time-of-Flight Mass Spectrometry for Compositional Profiling and Quantification of Heparin and Heparan Sulfate

Heparin and heparan sulfate (HS) are important pharmaceutical targets because they bind a large number of proteins, including growth factors and cytokines, mediating many biological processes. Because of their biological significance and complexity, there is a need for development of rapid and sensitive analytical techniques for the characterization and compositional analysis of heparin and HS at the disaccharide level, as well as for the structure elucidation of larger glycosaminoglycan (GAG) sequences important for protein binding. In this work, we present a rapid method for analysis of disaccharide composition using reversed-phase ion-pairing ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry ((RPIP)-UPLC-MS). Heparin disaccharide standards were eluted in less than 5 min. The method was used to determine the constituents of GAGs from unfractionated heparin/HS from various bovine and porcine tissues, and the results were compared with literature values.

Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10 min at ambient temperature) in the dark in sodium acetate buffer (80 mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150 mm × 4.6 mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was ≤1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7–101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140 pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were ≤0.2% of AA.

A new liquid chromatographic method for routine determination of oxytetracycline marker residue in the edible tissues of farm animals

A simple, rapid, and specific ion-pair liquid chromatographic method for routine determination of the marker residue of oxytetracycline (OTC), namely OTC and 4-epi-oxytetracycline (4-epiOTC), in edible animal tissues (muscle, liver, kidney, and fat) has been developed. Minced tissue samples were acidified at pH 2.7 with 2 mol L(-1) sulfuric acid and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulfate solution and concentrated into 0.1 mol L(-1) phosphoric acid. Baseline separation was carried out isocratically on a Nucleosil 100-5 C(18), 5-microm column using an acetonitrile-0.01 mol L(-1) disodium hydrogen phosphate (20:80, v/v) mobile phase that contained both positively (tetrabutylammonium) and negatively (octanesulfonate) charged pairing ions and EDTA, and was adjusted to pH 3.8. Detection was by UV at 370 nm. The method was fully validated according to Commission Decision 2002/657/EC. Overall recoveries were better than 82.6% and overall relative standard deviation was better than 6% for all the tissues examined. The good analytical characteristics of the method allowed limits of quantification as low as 30 ng g(-1) for muscle and fat and 50 ng g(-1) for liver and kidney, for both OTC and 4-epiOTC, to be realized. The method was successfully used to determine the OTC marker residue in tissues of two sheep intramuscularly administered a commercial OTC formulation.

Determination of N-Acetylcysteine and Thioglycolic Acid in Human Urine

A method for the determination of total N-acetylcysteine and thioglycolic acid in human urine is described. Because these compounds are mainly excreted as disulfides, they are first reduced to the free thiols by treatment with tris(2-carboxyethyl)phosphine hydrochloride and then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium derivatives of the thiols were achieved by reversed-phase ion-pair liquid chromatography with UV-detection at 355 nm. Because the method enables simultaneous determination of other endogenous urinary thiols, e.g. cysteine and cysteinylglycine, amounts of these compounds in urine were also studied. Detector responses were linear over the range covering most practical situations, with correlation coefficients for all four analytes better than 0.999. Recovery and imprecision (as RSD) were within 99.77–102.17 and 0.01–7.79%, respectively. The lower limit of detection was 0.25 μmol L−1 urine for thioglycolic acid and N-acetylcysteine, and 0.12 μmol L−1 urine for cysteine and cysteinylglycine. The method was used for analysis of urine samples from 29 healthy individuals to establish reference values for the thiols, normalized to creatinine. 3-Mercaptolactic acid, 2-mercaptopropionic acid, and mercaptoethanol were not present in the urine analyzed.

Simultaneous determination of amodiaquine and its active metabolite in human blood by ion-pair liquid chromatography–tandem mass spectrometry

A sensitive and selective ion-pair liquid chromatography–tandem mass spectrometric method (IP-LC–MS/MS) for the simultaneous determination of amodiaquine (AQ) and its active metabolite, N-desethylamodiaquine (AQm), in human blood has been developed and validated. Pentafluoropropionic acid (PFPA) was applied as ion-pairing reagent in reversed-phase chromatographic separation. The effects of PFPA concentrations and the volume fraction of acetonitrile in the mobile phase on the retention of analytes were investigated on a Venusil MP-C 18 column, and the mobile phase was finally optimized as acetonitrile:water (23:77, v/v) with 0.0667% PFPA in the aqueous phase. The results proved that PFPA as an ion-pairing reagent could provide desirable chromatographic performance in the IP-LC–MS/MS determination of 4-aminoquinoline compounds. Blood samples were protein precipitated with acetonitrile using hydroxychloroquine (OHCQ) as the internal standard. The detection was carried out in multiple reaction monitoring (MRM) mode via positive atmospheric pressure chemical ionization (APCI) interface. The lower limits of quantification were established at 0.150 and 1.50 ng/mL for AQ and AQm, respectively. The validated IP-LC–MS/MS method was applied to a clinical pharmacokinetic study of AQ and AQm in human blood after an oral administration of 600 mg AQ hydrochloride (459 mg base).

Validation of an ion-pair liquid chromatography–electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods

A method based on ion-pair liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS) is reported for determining heterocyclic aromatic amines (HAAs) in meat-based infant foods. The HAAs encompassed quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP), and carboline derivatives (AαC, Harman, Norharman) with d3-IQ, 13C2-MeIQx, and d3-PhIP used as labelled internal standards. The method used extraction into acetone followed by a clean-up on an SCX solid-phase extraction column. LC separation was performed on a TSKgel ODS-80TS column (250 × 2.0 mm, 5 µm), the mobile phase being an ammonium formate–formic acid buffer (3.03 mM ammonium formate, pH = 2.8) aqueous solution–acetonitrile gradient at a flow rate of 0.2 ml min−1. For unequivocal identification of each analyte, three ions were detected and chosen for selected reaction monitoring (SRM). Validation was carried out on lyophilized meat samples. Mean recoveries ranged between 78 ± 4% and 98 ± 2% for different analytes. Limits of quantification generally lower than 8 ng g−1 were demonstrated in meat samples for the analytes investigated. The method exhibited a good linearity and repeatability. Robustness testing identified those factors which were statistically significant in influencing chromatographic separation and response, and indicated which parameters have to be strictly controlled for a reliable analysis of HAAs. In particular, the mobile-phase flow rate was found to be statistically significant (α = 0.05) for the capacity factor (k′) of all analytes except for AαC peak, whereas the mobile-phase pH resulted to be a critical parameter for the k′ values of IQ, MeIQ, and Norharman. The method was proved to be robust vs. resolution between IQ and MeIQ peaks. Among mass-spectrometric parameters, collision energy was found to significantly affect quantitative response of all analytes except that of IQ. The applicability of the method to the analysis of meat-based infant food samples was demonstrated.

Application of Doehlert design in optimizing the determination of degraded products of nerve agents by ion-pair liquid chromatography electrospray ionization tandem mass spectrometry

A qualitative method was developed for the determination of degraded products of nerve agents by using ion-pair liquid chromatography electrospray ionization tandem mass spectrometry (IP-LC–ESI-MS n). Generally, alkylphosphonic acids (APAs) and O-alkyl alkylphosphonic acids (AAPAs) give deprotonated molecular ion [M−H] − in negative mode. Interestingly, first time we obtained the molecular radical anion [M] − of phosphonic acids in negative mode by using tri- n-butyl amine as an ion-pairing agent. We interpreted this observation as an indication of electrochemical reduction of phosphonic acids in electrospray needle. Three variables such as sheath gas flow, electrospray needle voltage and pH of the mobile phase were investigated to enhance the molecular radical anion [M] − signal of each analyte. The Doehlert design was used to obtain the region in which the optimum value of such variables is simultaneously achieved. Limit of detection achieved was 0.5 μg mL −1 for AAPAs and 10 μg mL −1 for APAs. Excellent precision was observed with less than 8.61% RSD. Finally, the method was applied for the detection of ethyl methylphosphonic in aqueous extract of soil sample.

Validated ion pair liquid chromatography/fluorescence detection method for assessing the variability of the loratadine metabolism occurring in bioequivalence studies

Inter- and intra-individual variability of the loratadine (LOR) metabolism in Caucasian subjects was assessed during a bioequivalence study for two pharmaceutical formulations (solid oral dosage forms) containing 10 mg of the active substance. The analytical data were obtained by applying a reliable, low-cost and sensitive ion pair liquid chromatography/fluorescence (IPLC/FLD) method for determination of both loratadine and descarboethoxyloratadine (DCL) in human plasma samples. The sample preparation procedure is based on liquid-liquid extraction of the target analytes from alkalinized plasma using diethyl-ether. The separation of the analytes and 8-chloroazatadine as internal standard (IS) was achieved through an isocratic ion pair (IP) elution on a Purospher((R)) STAR RP-18 column. The mobile phase containing sodium dodecyl sulfate (SDS) as ion pairing agent was pumped at a flow rate of 1 mL/min. Fluorescence detection (FLD) was achieved at 280 nm (excitation) and 440 nm (emission) wavelengths. The increased sensitivity of the method is also based on a large sample injected volume (250 microL). Linear response was found over the 0.5-20 ng/mL concentration interval for both target compounds. Low limits of quantification (LLOQ) around 0.3 ng/mL were found for LOR and DCL. Method validation is presented.

Determination of Chondroitin Sulfate Content in Raw Materials and Dietary Supplements by High-Performance Liquid Chromatography with Ultraviolet Detection After Enzymatic Hydrolysis: Single-Laboratory Validation

A method to quantify chondroitin sulfate in raw materials and dietary supplements at a range of about 5 to 100% (w/w) chondroitin sulfate has been developed and validated. The chondroitin sulfate is first selectively hydrolyzed by chondroitinase ACII enzyme to form un-, mono-, di-, and trisulfated unsaturated disaccharides; the resulting disaccharides are then quantified by ion-pairing liquid chromatography with ultraviolet detection. The amounts of the individual disaccharides are summed to yield the total amount of chondroitin sulfate in the material. Single-laboratory validation has been performed to determine the repeatability, accuracy, selectivity, limit of detection, limit of quantification, ruggedness, and linearity of the method. Repeatability precision for total chondroitin sulfate content was between 1.60 and 4.72% relative standard deviation, with HorRat values between 0.79 and 2.25. Chondroitin sulfate recovery from raw material negative control was between 101 and 102%, and recovery from finished product negative control was between 105 and 106%.

Chemical depolymerization of dextran sulfate sodium—the application of size‐exclusion or ion‐pair liquid chromatography

We reported previously a method for the separation and quantification of sulfated polysaccharides, dextran sulfate sodium (DSS), using fluorometric labeling and size-exclusion chromatography (SEC). In the present study, we evaluated chemical depolymerization of DSS. In fluorometric analyses using SEC, pyridylamino-DSS (PA-DSS) was depolymerized under acidic conditions, but not under alkaline conditions. In addition, we used ion-pair liquid chromatography (IPLC) and UV analyses for the separation and quantification of sulfate. Sulfate was considerably depleted from DSS under alkaline conditions, but little depleted under acidic conditions. Spectrophotometric studies using toluidine blue revealed that molecular mass is an important factor and a minimum molecular mass of 2500 is needed to induce metachromasia. Fluorometric analyses using SEC and UV analyses using IPLC allow for the detection of DSS, reactants and sulfate.

Quantitative determination of rosuvastatin in human plasma by ion pair liquid–liquid extraction using liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of rosuvastatin in human plasma. After being treated with acetic acid and tetrabutyl ammonium hydroxide, the analyte was extracted by simple one-step liquid–liquid extraction with the internal standard (IS: estrone). The chromatographic separation was performed on a Phenomenex Luna C 18 column with a mobile phase consisting of 2% formic acid/methanol (20:90, v/v) at a flow rate of 1.00 mL/min with a split of 200 μL to mass spectrometer. The retention time of rosuvastatin and internal standard was 2.3 and 3.4 min, respectively. Triple–quadrupole MS/MS detection was operated in positive mode by monitoring the transition of m/ z 482 → 258 for rosuvastatin and m/ z 271 → 253 for IS. Validation results indicated that the lower limit of quantification (LLOQ) was 0.1 ng mL −1 and the assay exhibited a linear range of 0.1–20 ng mL −1 and gave a correlation coefficient ( r) of 0.9990 or better. Inaccuracy was less than 8.4% and imprecision less than 12.8% at all tested concentration levels. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 8 weeks following storage at −20 °C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical pre-trials.

Liquid chromatography on an amide stationary phase with post-column derivatization and fluorimetric detection for the determination of streptomycin and dihydrostreptomycin in foods

The separation of streptomycin and its derivative dihydrostreptomycin using ion-pair liquid chromatography is proposed. The method is based on the use of a new stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl which avoids the appearance of tailed peaks. The isocratic mobile phase consisted of a 6:94 (v/v) acetonitrile/10 mM pentanesulfonic acid (pH 3.3) mixture at a flow-rate of 1 mL min −1 and fluorescence detection involved a post-column derivatization reaction using β-naphthoquinone-4-sulfonate. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of the aminoglycoside antibiotics in different types of foods, as honey, milk, egg and liver. Extraction was carried out by acidic hydrolysis to release protein-bound antibiotics. Detection limits in the food samples are 7.5 and 15 μg kg −1 for streptomycin and dihydrostreptomycin, respectively.

Liquids intermediate between “molecular” and “ionic” liquids: Liquid Ion Pairs?

Ionic liquids comprised of tetradecyltrihexyl- and tetrabutyl-phosphonium cations paired with chloride or sulfonyl amide anions exhibit properties that reflect strong ion association, including comparatively low viscosity as well as a degree of volatility, and hence exemplify an interesting intermediate state between true ionic and true molecular liquids.

Stability Indicating Methods for the Determination of Norfloxacin in Mixture with Tinidazole

Three stability indicating assay methods are developed for the determination of norfloxacin (Nor) in the presence of its decarboxylated degradation product and in mixture with tinidazole (Tnd). The proposed methods are reversed phase ion pair liquid chromatography (LC), thin layer densitometry (TLC) and second derivative ratio spectra zero crossing spectrophotometry ((2)DD). Chromatographic separation was achieved on mu-Bondapack C18 column 5 microm (300 mm x 3.9 mm, I.D.) and precoated silica gel TLC stationary phases for LC and TLC methods, respectively. Mobile phases consisting of phosphate buffer pH 3.2 : methanol (3 : 1, v/v) containing 0.005 M pentane sulfonic acid sodium salt and isopropanol : butanol : concentrated ammonia : water (25 : 50 : 5 : 25, v/v/v/v) were used for resolution of Nor and Tnd by both techniques, respectively. Detection was carried at 280 nm. In the ratio spectra method, detection of Nor was carried at 282 nm. Linearity, accuracy and precision were found to be acceptable over concentration ranges of 20-225 microg/ml, 0.8-4 microg/spot and 1-7 microg/ml for Nor by LC, TLC and (2)DD methods and over concentration ranges of 37.5-375 microg/ml and 4.8-20 microg/spot for Tnd by LC and TLC methods respectively. The suggested methods were successfully applied for the determination of both drugs in bulk powder, laboratory prepared mixtures and in commercial samples. Statistical comparison between the results obtained by the proposed and the reference methods was carried out using Student t-test, F ratio and one way ANOVA.

Simultaneous determination of Cr(iii) and Cr(vi) using reversed-phased ion-pairing liquid chromatography with dynamic reaction cell inductively coupled plasma mass spectrometry

A method for the simultaneous determination of Cr(III) and Cr(VI) species in waters, soil leachates and synthetic bio-fluids is described. The method uses reversed-phase ion-pairing liquid chromatography to separate the chromium species and a dynamic reaction cell (DRC®) equipped ICP-MS for detection of chromium. Separation of the chromium species is carried out in less than 2 min. Cr(III) is complexed with ethylenediaminetetraacetic acid (EDTA) prior to separation by mixing samples with the mobile phase containing 2.0 mM tetrabutylammonium hydroxide (TBAOH), 0.5 mM EDTA (dipotassium salt), and 5% (vol/vol) methanol, adjusted to pH 7.6. The interfering 40Ar12C+ background peak at mass 52 was reduced by over four orders of magnitude to less than 200 cps by using 0.65 mL min−1 ammonia as a reaction gas and an RPq setting on the DRC of 0.75. Method detection limits (MDLs) of 0.09 μg L−1 for Cr(III) and 0.06 μg L−1 for Cr(VI) were obtained based on peak areas at mass 52 for 50 μL injections of low level spikes. Reproducibility at 2 μg L−1 was 3% RSD for 5 replicate injections. The tolerance of the method to various levels of common cations and anions found in natural waters and to matrix constituents found in soil leachates and simulated gastric and lung fluids was tested by performing spike recovery calculations for a variety of samples.

Rapid arsenic speciation using ion pair LC-ICPMS with a monolithic silica column reveals increased urinary DMA excretion after ingestion of rice

Rapid separation of arsenic species is described by application of a commercial monolithic silica column (Chromolith™). Arsenic species were separated within 3 min, greatly reducing the time required with conventional columns. The separation was achieved by ion-pair liquid chromatography using 2.5 mM tetrabutylammonium bromide, 10 mM phosphate buffer (pH 5.6) and 1.0% (v/v) methanol as the mobile phase. Detection limits of 0.107, 0.084, 0.120, 0.121 and 0.101 μg L−1 As for As(III), arsenobetaine (AsB), dimethylarsinate (DMA), monomethylarsonate (MMA) and As(V), respectively were achieved. The precision of the method, based upon analysis of 15 μg L−1 As, was better than 5.9% for all species. The technique was applied to the speciation analysis of urine and food samples in an ingestion experiment to investigate the effect of rice consumption on arsenic exposure in humans. For the first time this study reveals that ingestion of American long grain rice, which is widely consumed in the UK and purchased from a local supermarket, significantly increases the excretion of DMA in human urine. This is consistent with a recent study which reported that DMA was the predominant arsenic species in rice from the USA, which also happened to contain the highest mean arsenic level in the grain compared to rice grown in Bangladesh and India.1

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD ⩽0.7 ng on-column), repeatable ( σ rel ⩽ 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation ⩽9.4%, interday variation ⩽6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 °C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.