Analysis of lipids from crude lung tissue extracts by desorption electrospray ionization mass spectrometry and pattern recognition

A method is described using desorption electrospray ionization (DESI) mass spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mouse lungs. Specifically, the two vaccination routes compared were intranasal (i.n.) and intradermal (i.d.) inoculation of the Francisella tularensis live vaccine strain (Ft–LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution, and the crude extract was directly analyzed by DESI–MS, with a total turnaround time of less than 10 min/sample. All of the measured DESI mass spectra (in both positive and negative ion mode) were compared via PCA, resulting in clear differentiation of mass spectral profiles of i.n.-inoculated mouse lung tissues from those of i.d.-inoculated and control mouse lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI–MS approach described here provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Simultaneous quantitation of perfluoroalkyl acids in human serum and breast milk using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

A high throughput analytical method using a column switching high-performance liquid chromatography combined with isotope dilution tandem mass spectrometry (column switching-HPLC–MS/MS) was developed to simultaneously quantitate the concentrations of 7 perfluoroalkyl acids (PFAAs) in serum and 3 PFAAs in breast milk samples. The sample preparation includes addition of the isotope-labelled internal standard solution to breast milk and serum, enzymatic hydrolysis and filtration of milk samples, precipitation of proteins and analysis by column switching-HPLC–MS/MS. The limits of quantitation ranged from 0.1 to 0.4 μg/l for serum and 0.02 to 0.15 μg/l for breast milk samples. The method accuracies ranged between 73.2% and 100.2% for the different analytes at two concentrations in PFAAs spiked samples. The validity of the method was confirmed by analysing 20 serum and 20 breast milk samples.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Pitfalls in the sample preparation and analysis of N-acylethanolamines

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.     

Urinary organic acid screening by solid-phase microextraction of the methyl esters

We developed a new sample preparation method for profiling organic acids in urine by GC or GC–MS. The method includes derivatisation of the organic acids directly in the aqueous urine using trimethyloxonium tetrafluoroborate as a methylating agent, extraction of the organic acid methyl esters from the urine by solid-phase microextraction, using a polyacrylate fiber with a thickness of 85 μm and transfer of the methyl esters into the GC or the GC–MS instrument. Desorption of the analytes takes place in the heated injection port. The proposed sample preparation is very simple. There is no need for any evaporation step and for the use of an organic solvent. The risk of contamination and the loss of analytes are minimized. The total sample preparation time prior to GC or GC–MS analysis is about 40 min, and therefore more rapid than other sample preparation procedures. The urinary organic acids are well separated by GC and 29 substances are identified by GC–MS.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.     

Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Optimizing sequence coverage for a moderate mass protein in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI–Q-TOF–MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). The sample pretreatment/nESI–Q-TOF–MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.     

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.     

Oxidized fatty acid analysis by charge-switch derivatization,selected reaction monitoring,and accurate mass quantitation

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0 pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r² > 0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA₂γ^–/– mouse liver identifying the role of iPLA₂γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.     

Quantitative analysis of adenosine using liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS)

Adenosine-secreting cellular brain implants constitute a promising therapeutic approach for the treatment of epilepsy. To engineer neural stem cells for therapeutic adenosine delivery, a reliable and fast analytical method is necessary to quantify cell-based adenosine release. Here we describe the development, optimization and validation of adenosine measurement using liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC–APCI-MS/MS). LC–MS/MS in positive ion mode used selected reaction monitoring at m/z of 268.2/136.1 and 302.2/170.0 for adenosine and the internal standard, respectively. The bias was within 15% of the nominal value and evaluation of precision showed a relative standard deviation lower than 15% for all measured concentrations. The lower limit of quantification of adenosine was 15.6 ng/ml. Freeze and thaw stability and processed sample stability also fulfilled the acceptance criteria. Evaluation of the matrix effect showed that the method is not affected by relative matrix effects. The major advantages of this method are the absence of an extraction phase and the combination of the high selectivity and sensitivity characteristic for the LC–MS/MS technique, with a short run time of 4.5 min. These results demonstrate that this method is a useful tool to measure adenosine concentrations in culture medium released from stem cells in vitro.     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4-¹³C¹⁵N₂ as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.     

GC–MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC–UV, GC–MS, and LC–MS and LC–MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC–MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d₀-Crea) and the internal standard [methyl-trideutero]creatinine (d₃-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d₀-Crea-PFB and m/z 115 for d₃-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC–MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC–MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC–MS method. The application of the GC–MS method can be extended to other biological samples such as saliva.