Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d₃ and 7-AF-d₃ were used as internal standards. The drugs were separated on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d₃ and F-d₃), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d₃), 330 (3-OHF). The limits of detection were: 0.2 μg/l for F and 7-AF, 1 μg/l for N-DF and 3-OHF. The method was linear in the range 1–500 μg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC–APCI-MS seems to be a method of choice for these compounds.     

Class-specific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography–tandem mass spectrometry

A novel method based on the molecularly imprinted solid-phase extraction (MISPE) procedure has been developed for the simultaneous determination of concentrations of sulfonylurea herbicides such as chlorsulfuron (CS), monosulfuron (MNS), and thifensulfuron methyl (TFM) in maize samples by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). The molecularly imprinted polymer (MIP) for sulfonylurea herbicides was synthesized by precipitation polymerization using chlorsulfuron as the template molecule, 2-(diethylamino)ethyl methacrylate (DEAMA) as the functional monomer, and trimethylolpropane trimethacrylate (TRIM) as the cross-linker. The selectivities of the chlorsulfuron template and its analogs on the molecularly imprinted polymer were evaluated by high-performance liquid chromatography (HPLC). The extraction and purification procedures for the solid-phase extraction (SPE) cartridge with a molecularly imprinted polymer as the adsorbent for the selected sulfonylurea herbicides were then established. A molecularly imprinted solid-phase extraction method followed by high-performance liquid chromatography–tandem mass spectrometry for the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl was also established. The mean recoveries of these compounds in maize were in the range 75–110% and the limits of detection (LOD) of chlorsulfuron, monosulfuron, and thifensulfuron methyl were 0.02, 0.75, and 1.45 μg kg⁻¹, respectively. It was demonstrated that the MISPE–HPLC–MS/MS method could be applied to the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl in maize samples.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography

Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.     

Rapid determination of methadone and its major metabolite in biological fluids by gas–liquid chromatography with thermionic detection for maintenance treatment of opiate addicts

A rapid gas–liquid chromatographic assay is developed for the quantification of methadone (Mtd) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in biological fluids of opiate addicts. After alkaline extraction from samples with lidocaine hydrochloride as internal standard, Mtd and EDDP are separated on SP-2250 column at 220°C and detected with a thermionic detector. The chromatographic time is about 6 min. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 1.5 and 5.5%. Most drugs of abuse (morphine, codeine, narcotine, cocaine, benzoylecgonine, cocaethylene, dextropropoxyphene etc) are shown not to interfere with this technique. The method has been applied to study the levels of Mtd and EDDP metabolite in serum, saliva and urine of patients under maintenance treatment for opiate dependence. EDDP levels were found higher than those of Mtd in urine samples from four treated patients, but lower in serum and undetectable in saliva. However, Mtd concentrations were higher in saliva than in serum.     

LC–MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma

A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150 mm × 4.6 mm, 3 μm) column using a gradient elution mode with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10 mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68–106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.     

Characterization of monoclonal antibodies by a fast and easy liquid chromatography–mass spectrometry time-of-flight analysis on culture supernatant

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain mass spectrometry profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates three fragments of 25 kDa that can be analyzed by liquid chromatography–mass spectrometry time-of-flight (LC–MS TOF) in one run: LC, Fd, and Fc/2. This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process was successfully compared with high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE–LIF), currently considered as the “gold standard” method.     

Measurement of pyrethroid,organophosphorus, and carbamate insecticides in human plasma using isotope dilution gas chromatography–high resolution mass spectrometry

We have developed a gas chromatography–high resolution mass spectrometry method for measuring pyrethroid, organophosphorus, carbamate and fipronil pesticides and the synergist piperonyl butoxide in human plasma. Plasma samples were extracted using solid phase extraction and were then concentrated for injection and analysis using isotope dilution gas chromatography–high resolution mass spectrometry. The limits of detection ranged from 10 to 158 pg/mL with relative recoveries at concentrations near the LODs (e.g., 25 or 250 pg/mL) ranging from 87% to 156% (9 of the 16 compounds were within ±15% of 100%). The extraction recoveries ranged from 20% to 98% and the overall method relative standard deviations were typically less than 20% with some exceptions. Analytical characteristics were determined at 25, 250, and 1000 pg/mL.     

Smelling the Diagnosis: The Electronic Nose as Diagnostic Tool in Inflammatory Arthritis. A Case-Reference Study

Objective

To investigate whether exhaled breath analysis using an electronic nose can identify differences between inflammatory joint diseases and healthy controls.

Methods

In a cross-sectional study, the exhaled breath of 21 rheumatoid arthritis (RA) and 18 psoriatic arthritis (PsA) patients with active disease was compared to 21 healthy controls using an electronic nose (Cyranose 320; Smiths Detection, Pasadena, CA, USA). Breathprints were analyzed with principal component analysis, discriminant analysis, and area under curve (AUC) of receiver operating characteristics (ROC) curves. Volatile organic compounds (VOCs) were identified by gas chromatography and mass spectrometry (GC-MS), and relationships between breathprints and markers of disease activity were explored.

Results

Breathprints of RA patients could be distinguished from controls with an accuracy of 71% (AUC 0.75, 95% CI 0.60–0.90, sensitivity 76%, specificity 67%). Breathprints from PsA patients were separated from controls with 69% accuracy (AUC 0.77, 95% CI 0.61–0.92, sensitivity 72%, specificity 71%). Distinction between exhaled breath of RA and PsA patients exhibited an accuracy of 69% (AUC 0.72, 95% CI 0.55–0.89, sensitivity 71%, specificity 72%). There was a positive correlation in RA patients of exhaled breathprints with disease activity score (DAS28) and number of painful joints. GC-MS identified seven key VOCs that significantly differed between the groups.

Conclusions

Exhaled breath analysis by an electronic nose may play a role in differential diagnosis of inflammatory joint diseases. Data from this study warrant external validation.     

High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography–mass spectrometry (LC–MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2–5 μg of protein. The method is also compatible with our standard LC–MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.     

Hair testing for drugs of abuse

Hair testing for drugs of abuse is a developing technology, which offers the possibility of longer detection times than is commonly obtained with urine analysis. It is the main method for evaluation of an individual's drugs of abuse history. In many countries hair analysis is routinely used to detect drug abuse in forensic cases, occupational and traffic medicine and clinical toxicology. Hair analysis in pregnant women, neonates and infants is a useful tool for the detection of drug exposure in utero. In Croatia hair testing for drugs of abuse is performed at the Institute for Medical Research and Occupational Health. Three-year experience in drugs of abuse analysis in hair is described. In 331 hair samples (270 from adolescents and 61 from adults) opiates and metabolites, cocaine, methadone, and amphetamines were analyzed by gas chromatography/mass spectrometry. Most prevalent drugs of abuse in adolescents were amphetamines, and in adults heroin. From the examples cited and samples analyzed it is evident that hair testing is emerging as a reliable biological marker for cumulative account of individual exposure to drugs of abuse.     

Sensitive determination of methomyl in blood using gas chromatography–mass spectrometry as its oxime tert.-butyldimethylsilyl derivative

A sensitive, selective and reliable method was developed to determine methomyl {methyl-N-[(methylcarbamoyl)oxy]thioacetimidate}, a carbamate insecticide in human blood, using gas chromatography–mass spectrometry. Dimethylglyoxime served as an internal standard (I.S.). Methomyl in the blood was converted to its oxime form by sodium hydroxide. The solution made acidic with hydrochloric acid was poured into a column packed with Extrelut. Methomyloxime and I.S. were eluted from the column with a mixture of dichloromethane–ethyl acetate–chloroform (65:25:10), transformed to tert.-butyldimethylsilyl derivatives, and analyzed by gas chromatography–mass spectrometry in the electron impact mode. The calibration curves were linear in the concentration range from 1 ng/g to 100 ng/g and 100 ng/g to at least 5000 ng/g. The lower limit of detection was 0.5 ng/g. The absolute recoveries were 72–93% and within-day coefficients of variation were 3.1–5.6% at blood concentrations of 10 and 1000 ng/g. Two practical forensic applications are described.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Determination of proteomic and metabolic composition of exhaled breath condensate of newborns

Here, the possibility of proteomic and metabolomic analysis of the composition of exhaled breath condensate of neonates with respiratory support. The developed method allows non-invasive collecting sufficient amount of the material for identification of disease-specific biomarkers. Samples were collected by using a condensing device that was incorporated into the ventilation system. The collected condensate was analyzed by liquid chromatography coupled with high resolution mass spectrometry and tandem mass spectrometry. The isolated substances were identified with a use of databases for proteins and metabolites. As a result, a number of compounds that compose the exhaled breath condensate was determined and can be considered as possible biomarkers of newborn diseases or stage of development.     

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography–mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3 h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography–tandem mass spectrometry method.     

Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.