Measurement of total and free malondialdehyde by gas–chromatography mass spectrometry – comparison with high-performance liquid chromatography methology

Abstract

Malondialdehyde (MDA) is considered to be a biomarker for enzymatic degradation and lipid peroxidation of polyunsaturated fatty acids. Usually, MDA determination from different biological materials is performed by reaction with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis and fluorometric detection. As this method lacks specificity and sensitivity, we developed a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of MDA with 2,4-dinitrophenylhydrazine. Representative ions in negative ion chemical ionization (NICI) mode were recorded at m/z 204 for MDA and at m/z 206 for the deuterated analogon (MDA-d₂) as internal standard. This stable and precise GC–MS method showed good linearity (r² = 0.999) and higher specificity and sensitivity than the HPLC method and was validated for both total MDA (t-MDA) and free MDA (f-MDA). Within-day precisions were 1.8–5.4%, between-day precisions were 4.8–9.2%; and accuracies were between 99% and 101% for the whole calibration range (0.156–5.0 μmol/L for t-MDA and 0.039–0.625 μmol/L for f-MDA). Although comparison of t-MDA levels from GC–MS and HPLC results using Passing–Bablok regression analysis as well as Bland–Altman plot showed a correlation of the data, a tendency to increased results for the HPLC values was detectable, due to possible formation of unspecific products of the TBA reaction.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid–liquid extraction, and analyzed by LC–ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Investigations on the biodegradation of alkylpolyglucosides by means of liquid chromatography–electrospray mass spectrometry

The biodegradation of alkylpolyglucosides (APGs) was studied under the conditions of the OECD Screening Test with activated sludge as an inoculum. An influence of alkyl and sugar chain length on the biodegradation rate and a central scission pathway of the biodegradation were investigated. The liquid chromatography-electrospray mass spectrometry technique was used for alkylpolyglucoside analysis and for identification and semiquantitative determination of metabolites. It was found that APGs with a longer alkyl chain were biodegraded faster than those with a shorter one. However, a longer sugar chain caused slower biodegradation of APGs. The central scission pathway of biodegradation was also confirmed.     

Quality evaluation of Semen persicae (seed of Prunus persica L. Batsch) by gas chromatography–mass spectrometry

Seeds of Prunus persica L. Batsch were collected at various times from different regions of China. The fatty acid composition of the seeds was analysed using gas chromatography-mass spectrometry (GC-MS). The aim was to evaluate whether genetic and environmental factors influenced the chemical profile of fatty acids in Semen persicae. Saturated and unsaturated fatty acids were present at 4.8-8.7% and 90.7-94.8% of total fatty acids, respectively. Oleic and linoleic acids were dominant in all samples and ranged from 59.3 to 81.4% and from 11.6 to 31.0%, respectively. All samples had high levels of unsaturated fatty acids. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples based on the contents of the characteristic fatty acid constituents. This study provides evidence that metabolites may reflect genetic and environmental similarities, such as management practices used in cultivars (irrigation, fertilization and sanitary treatments), and evidence of the variability of genetic make up and local environmental conditions in samples grown in the wild.     

Optimized analytical method for cyclosporin A by high-performance liquid chromatography–electrospray ionization mass spectrometry

The Micromass Platform LCZ mass detector parameters were optimized for simultaneous recording of the protonated (CsA∼H⁺), sodium adduct (CsA∼Na⁺) and potassium adduct (CsA∼K⁺) of cyclosporin A eluted from a Symmetry Shield RP8 column. The optimized procedure allows a precise analysis of CsA in whole blood or serum without removal of salts prior to analysis. The ratio of the three forms of CsA varied depending on the assay condition and the types of specimens being analyzed. The summation of three ionic forms of CsA detected by LC–ESI-MS is a reliable and simple method to assess CsA concentration in the blood.     

Analysis of corticosteroids in equine urine by liquid chromatography–mass spectrometry

A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with β-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC–MS and confirmed by LC–MS–MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.     

Simultaneous measurement of aldosterone and cortisol by high-performance liquid chromatography–tandem mass spectrometry: Application to dehydration–rehydration studies

Aldosterone and cortisol are useful biomarkers of dehydration and stress, respectively. The aim of this study was to develop an HPLC–tandem mass spectrometric method for the simultaneous measurement of aldosterone and cortisol in human plasma that could be applied to the study of athletes undergoing exercise and rehydration. Samples were prepared and analysed using an on-line sample preparation/HPLC system coupled to a triple quadrupole tandem-mass spectrometer. Samples (200 μL) were pre-treated and extracted on Hysphere C18 HD cartridges (7 μm, Spark Holland). Chromatography was performed on a Sunfire C18 analytical column (50 mm × 3.0 mm, 3 μm, Waters) under isocratic conditions at a flow rate of 0.3 mL/min. The mobile phase consisted of 35% acetonitrile/water. Mass spectrometric detection was by selected reaction monitoring using negative electrospray ionization conditions. The assay had an analytical range of 25–500 pg/mL and 25–500 ng/mL for aldosterone and cortisol, respectively (r² > 0.992, n = 22). Inter-day accuracy and imprecision for quality control samples was 99.4–106% and <16%, respectively (n = 10). In a study of nine human subjects, both aldosterone and cortisol concentrations reflected the expected physiological responses to dehydration, rehydration and exercise when measured by this method. The reported method is suitable to facilitate the study of athletes undergoing dehydration and rehydration protocols.     

Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography–mass spectrometry

A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.     

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r² > 0.997, n = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n = 5) and inter-day (n = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

Simultaneous detection of residues of β-adrenergic receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass spectrometry

A method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was developed to screen and confirm residues of multi-class veterinary drugs in animal tissues (porcine kidney, liver, muscle; bovine muscle). Thirty target drugs (19 β-blockers, 11 sedatives) were determined simultaneously in a single run. Homogenized tissue samples were extracted with acetonitrile and purified using a NH₂ solid-phase extraction cartridge. An Acquity UPLC? BEH C18 column was used to separate the analytes, followed by tandem mass spectrometry using an electrospray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries in pig muscle, kidney, and liver fortified at three levels from 76.4% to 118.6% based on matrix-fortified calibration with coefficients of variation from 2.2% to 19.9% (n = 6). The limit of quantification of these compounds in different matrices was 0.5–2.0 μg/kg. This method was successfully applied in screening and confirming target drugs in >200 samples.     

Identification of flavanone metabolites in rat urine by combined gas–liquid chromatography and mass spectrometry

1. The metabolism of flavanone in the rat was studied after oral or intraperitoneal administration of the compound. Flavone and flav-3-ene together with five other unidentified minor metabolites were excreted in the urine. 2. The formation of flavanone metabolites was not suppressed by the administration of high doses of the antibacterial compounds aureomycin and phthaloylsulphathiazole. 3. No aromatic acids that could be attributed to ring cleavage of flavanone were detected. 4. Administration of 100 or 200mg of flavanone daily per rat caused some deaths during the 7-14-day period. 5. The application of combined gas-liquid chromatography/mass spectrometry and proton nuclear-magnetic-resonance spectroscopy to the separation and identification of the flavanone metabolites is described. 6. Measurement of the two major flavanone metabolites was carried out by gas-liquid chromatography.     

A high-performance liquid chromatography–mass spectrometry assay for quantitation of the tyrosine kinase inhibitor nilotinib in human plasma and serum

Nilotinib (AMN-107, Tasigna?) is a small-molecule inhibitor of BCR/ABL, approved for chronic myelogenous leukemia. We developed and validated, according to FDA-guidelines, an LC–MS assay for sensitive, accurate and precise quantitation of nilotinib in 0.2 mL human plasma or serum. After acetonitrile protein precipitation, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in methanol/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry. Between 5 (LLOQ) and 5000 ng/mL, accuracy (92.1–109.5%), intra-assay precision (2.5–7.8%), and inter-assay precision (0–5.6%)) were within FDA limits. We demonstrated the suitability of this assay by quantitating plasma concentrations of nilotinib in a healthy volunteer after oral administration of 400 mg nilotinib.     

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.