Modelling nicotine intake in smokers and snuff users using biological fluid nicotine metabolites

Data from two clinical studies involving smokers and snuff users were analysed to address the estimation of nicotine intake using urinary and salivary nicotine metabolites. Comprehensive regression modelling is performed to determine which combinations of urinary nicotine metabolites provide better estimation of nicotine intake in these subjects than the predominant practice of basing nicotine intake on urinary cotinine analysis alone. Within-subject and between-subject variability is examined with regard to reliability of measurement and replicate sampling. Salivary cotinine models are compared to urinary metabolite models. Results suggest that estimation of nicotine intake is greatly improved by measuring urinary cotinine and additional metabolites (trans-3´-hydroxycotinine, and glucuronide conjugates) rather than measuring only cotinine. Analyses also indicate that replicate sampling on subjects greatly improves the reliability of the measurement. Based on these data, a model to predict nicotine equivalents based solely on saliva cotinine was severely inferior to any of the urinary models, including that of urinary cotinine alone.     

Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) assay for the determination of nicotine and cotinine in human milk was developed using an extraction by liquid-liquid partition combined with back extraction into acid, and followed by reverse-phase chromatography with UV detection of analytes. The assay was linear up to 500 microg/l for both nicotine and cotinine. Intra- and inter-day relative standard deviations (R.S.D.) were <10% (25-500 microg/l) for both nicotine and cotinine. Limits of quantitation (LOQ) were 10 and 12 microg/l for nicotine and cotinine, respectively, while the limits of detection (LOD) were 8 and 10 microg/l for nicotine and cotinine, respectively. The mean recoveries were 79-93% (range 25-500 microg/l) for nicotine and 78-89% (range 25-500 microg/l) for cotinine. The amount of fat in the milk did not affect the recovery. We found that this method was sensitive and reliable in measuring nicotine and cotinine concentrations in milk from a nursing mother who participated in a trial of the nicotine patch for smoking cessation.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Rapid method for the simultaneous measurement of nicotine and cotinine in urine and serum by gas chromatography–mass spectrometry

A simple, sensitive, and rapid gas chromatographic–mass spectrometric method is described for the simultaneous detection and quantitation of nicotine and its metabolite, cotinine, in urine and serum. The analytes and their respective deuterated internal standards were extracted by liquid–liquid extraction coupled to centrifugation and evaporation. The detection limit of the assay was 0.16 ng/ml for both nicotine and cotinine. The limit of quantitation for each analyte was 1.25 ng/ml.     

Novel liquid chromatographic–tandem mass spectrometric methods using silica columns and aqueous–organic mobile phases for quantitative analysis of polar ionic analytes in biological fluids

Use of silica stationary phase and aqueous–organic mobile phases could significantly enhance LC–MS–MS method sensitivity. The LC conditions were compatible with MS detection. Analytes with basic functional groups were eluted with acidic mobile phases and detected by MS in the positive ion mode. Analytes with acid functional groups were eluted with mobile phases at neutral pH and detected by MS in the negative ion mode. Analytes poorly retained on reversed-phase columns showed good retention on silica columns. Compared with reversed-phase LC–MS–MS, 5–8-fold sensitivity increases were observed for basic polar ionic compounds when using silica columns and aqueous–organic mobile phase. Up to a 20-fold sensitivity increase was observed for acidic polar ionic compounds. Silica columns and aqueous–organic mobile phases were used for assaying nicotine, cotinine, and albuterol in biological fluids.     

Determination of nicotine and cotinine in human serum by means of LC/MS

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.     

Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection

Nicotine can be determined in pharmaceuticals and biological fluids by micellar liquid chromatography (MLC) using a C18 column, a mobile phase containing sodium dodecyl sulphate 0.15 M–6% (v/v) pentanol–NaH₂PO₄ 0.01 M (pH 6)–KCl 0.001 M, with electrochemical detection at 0.8 V. In the optimization step, the influence of the modifiers propanol, butanol and pentanol, and the voltage has been studied. With the proposed method the analysis time is below than 8 min, linearity better than 0.999, limits of detection and quantification (ng/ml) was 4 and 12 respectively, repeatability and intermediate precision below 1.8%, and all these parameters are adequate for the quantification of nicotine in chewing gum, dermal patches, tobacco and serum samples either by a pharmacologist, pathologist or toxicologist.     

Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry

A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N-oxide, norcotinine, and nicotine-N'-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3'-hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC-NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N-glucuronide (coefficient of variation, CV: 9%) and trans-3'-hydroxycotinine-O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC-MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.     

Profile of urinary phenanthrene metabolites in smokers and non-smokers

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.     

Profile of urinary phenanthrene metabolites in smokers and non-smokers

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.     

Validation of an assay for the determination of cotinine and 3-hydroxycotinine in human saliva using automated solid-phase extraction and liquid chromatography with tandem mass spectrometric detection

The validation of a high-performance liquid chromatographic method for the simultaneous determination of low level cotinine and 3-hydroxycotinine in human saliva is reported. Analytes and deuterated internal standards were extracted from saliva samples using automated solid-phase extraction, the columns containing a hyper cross-linked styrene–divinylbenzene copolymer sorbent, and analysed by reversed-phase liquid chromatography with tandem mass spectrometric detection (LC–MS–MS). Lower limits of quantitation of 0.05 and 0.10 ng/ml for cotinine and 3-hydroxycotinine, respectively, were achieved. Intra- and inter-batch precision and accuracy values fell within ±17% for all quality control samples, with the exception of quality control samples prepared at 0.30 ng/ml for 3-hydroxycotinine (inter-day precision 21.1%). Results from the analysis of saliva samples using this assay were consistent with subjects’ self-reported environmental tobacco smoke (ETS) exposures, enhancing the applicability of cotinine as a biomarker for the assessment of low level ETS exposure.     

Good results of circumferential spine fusion in smokers, using autograft and allograft

We have examined the rates of anterior interbody fusion of lumbar spine segments following fusion with a fresh frozen femoral head allograft in 25 heavy smoking patients. They were all stabilized both anteriorly and posteriorly. The indications for surgery were: degenerative disc disease, degenerative spondylolisthesis and nonunion following previous posterolateral fusion of lumbar spine segments. Only patients who had fusion of one or two lumbar segments were included. They all were stabilized posteriorly with pedicle screws and autogenic iliac bone graft. The fusion was assessed at least one year after surgery according to plan X-rays as “Solid”, “Questionable” or “Failure”. One patient was found at follow up not fused, in another one the fusion was “questionable” and all the other 23 patients had an anterior solid fusion. Clinically, 84% of the patients had the same or improved work status as before surgery and 68% acknowledged that they were satisfied with the surgical results. No major complications were recorded and the average length of hospitalization was 10.3 days. This revised version was published online in July 2006 with corrections to the Cover Date.     

Predicting medicinal resources in Ranunculaceae family by a combined approach using DNA barcodes and chemical metabolites

Plant taxonomy based on molecular phylogenetic study and/or chemosystematics study has become increasingly important in exploring and utilizing medicinal resources due to the advent of big data era. In this study, we proposed a classifying approach combining DNA and chemical metabolites for the prediction of new medicinal resources. Specifically, we obtained 104 ITS2 barcodes and 847 chemical metabolites from 104 species in Ranunculaceae. Then, phylogenetic tree based on the ITS2 barcode and clustering tree based on structural similarity of metabolites were separately constructed. In addition, we tested the classifying accuracy of the two methods by Baker`s correlation coefficient and the result showed that phylogenetic tree based on the ITS2 barcode was more accurate, giving a higher score of 0.627, whereas clustering tree based on chemical metabolites obtained a lower score of 0.301. Therefore, the natural products of plants might be described using these clades found by ITS2-based methods, and thus new metabolites of plants might be predicted due to the close relationships in a given clade. Using this combined method, 53 plants with structurally similar metabolites were included in 9 plant groups and currently unknown species-metabolite relations were predicted. Finally, 26.92% species in Ranunculaceae were found to contain the predicted metabolites after verification using two alternative KNApSAcKCore and ChEBI databases. As a whole, the combined approach can successfully classify plants and predict specialized natural products based on plant taxa.     

Quantification of PPAR-gamma protein in monocyte/macrophages from healthy smokers and non-smokers: a possible direct effect of nicotine

Previous observations demonstrated that Peroxisome Proliferator-Activated Receptor-gamma (PPAR-gamma), a key regulator of adipocyte differentiation, is expressed in a large variety of cells, including cells of the monocyte/macrophage lineage. This study was aimed to quantify both the constitutive and ligand-induced PPAR-gamma expression in monocytes and monocyte-derived macrophages (MDM) isolated from healthy smokers and non-smokers, and to evaluate the possible direct effect of nicotine. PPAR-gamma protein was detected by Western blot and quantification was performed by calculating the ratio between PPAR-gamma and beta-actin protein expression. Cytokine release was measured with enzyme-linked immunoassay kits. Constitutive PPAR-gamma protein was detected in human monocytes and its expression was up-regulated along with differentiation to MDM. The endogenous ligand 15-deoxy-delta(12,14)-prostaglandin J(2) and the synthetic agonist ciglitazone enhanced PPAR-gamma expression, the former being effective also at low micromolar concentrations. Both agonists significantly inhibited the basal secretion of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6), ciglitazone being more potent. Monocytes and MDM from healthy smokers presented a significantly enhanced (4-fold and 2.5-fold, respectively) constitutive PPAR-gamma expression, as compared to those from healthy non-smokers. However, ligand-induced PPAR-gamma expression and inhibition of cytokine secretion were similar in healthy smokers and non-smokers. Nicotine dose-dependently enhanced PPAR-gamma expression with a maximum at 10 muM, and inhibited release of pro-inflammatory cytokines; these effects were reversed by alpha-bungarotoxin. Nicotine and PPAR-gamma agonists did not exert synergistic effects. In conclusion, monocytes and MDM from healthy smokers present a constitutively enhanced PPAR-gamma expression; this effect is reproduced, to some extent, by nicotine in vitro.     

Review on the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its phase I and phase II metabolites in biological matrices, foodstuff and beverages

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), has been shown to be carcinogenic in rodents, mice and rats. Following phase I N-hydroxylation and phase II esterification PhIP exerts its carcinogenic effect by binding to DNA purines. Quantitative and qualitative analysis of its bioactivated metabolites as well as it detoxification products is important in studying its biological effects and inter- and intra-individual exposures. A review is presented with an extensive coverage of publications specifically reporting on the analysis of PhIP and its phase I and II metabolites in biological matrices, foodstuff and beverages. Analytical techniques such as liquid and gas chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection) were mostly applied. We conclude that since the initial identification of PhIP in 1986 a large set of assays has been developed for the analysis of PhIP and its phase I and phase II metabolites in a wide range of matrices, these included food products and biological samples such as plasma, urine and faeces. In addition, it was shown that numerous metabolites were recovered and identified. Thus, we conclude that liquid chromatography coupled to mass spectrometry is clearly the method of choice for sensitive qualitative as well as quantitative analysis with high selectivity and reaching lower quantification levels in the sub pg/mL range. The main aim of this review is that it can be used by other researchers as a resource for method development and optimization of analytical methods of PhIP and its carcinogenic or detoxification products.     

Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1–85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2±7.9, 74.9±8.5, 81.7±6.9, 73.1±7.8, 74.3±8.3, 80.8±6.6, 81.6±7.3 and 81.4±6.5, and from urine 79.4±6.9, 77.8±8.4, 83.3±6.6, 72.8±9.0, 76.3±7.7, 83.4±7.9, 81.6±7.9 and 81.8±6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB.     

Determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3'-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.     

Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design

As a basis for the development of predictive mathematical models in systems biology and a quantitative understanding of cellular metabolism, reliable experimental data sets of intracellular metabolites are indispensable. A prerequisite for the acquisition of such data is the identification of a suitable sample preparation method. In this work, the extraction procedure for the simultaneous measurement of a wide range of intracellular metabolites from adherent mammalian cells in culture was optimized. A screening of several commonly used extraction protocols with Madin-Darby canine kidney (MDCK) cells found the methanol/chloroform (MeOH/CHCl(3)) and MeOH/Boil methods to be promising candidates for further analysis by anion-exchange chromatography. Both methods were optimized based on experimental design techniques with four response variables: Nucleotide Content, Energy Charge, Fructose 1,6-Bisphosphate content (F16bP), and Absorption at 280 nm. After data evaluation and with the help of desirability functions, an overall optimum for the extraction conditions was found. Using optimal settings, the extraction performances for MDCK and Vero cell cultivations of both methods were compared. Both methods extracted nearly the same absolute amounts of intracellular metabolites, suggesting that these methods are equal. However, recoveries for nucleotide diphosphates were significantly above 100% for both methods. This most likely was due to remaining nucleotide kinase activity during extraction. After combining individual steps of both methods, recoveries close to 100% for all metabolites could be reached. Absolute values of intracellular metabolites extracted with this modified method are comparable to the results of the two previously optimized methods, indicating a good extraction procedure according to the chosen response variables.