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.     

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography–tandem mass spectrometry

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 μm 50 mm × 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 μg/ml when 100 μl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C_max) 0.40, 0.57, 1.95 μg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31 μg/ml on Day 28 for low, mid and high dose treated animals.     

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap-tandem mass spectrometry coupled with electro spray ionization (LC–ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C₁₈ ACE^® column (50.0 mm × 2.1 mm, 5 μm, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3 mL/min. The transition monitored (positive mode) was m/z 279.1 → m/z 208.1 for triprolidine, m/z 165.9 → m/z 148.0 for pseudoephedrine and m/z 172.0 → m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0 min and a linear calibration curves ranged from 0.2 to 20.0 ng/mL for triprolidine and 5.0–500.0 ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3–106.3% and 1.0–9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.     

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).     

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.     

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.     

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.     

Determination of cymipristone in human plasma by liquid chromatography–electrospray ionization-tandem mass spectrometry

A rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for determination of cymipristone in human plasma. Mifepristone was used as the internal standard (IS). Plasma samples were deproteinized using methanol. The compounds were separated on a ZORBAX SB C₁₈ column (50 mm × 2.1 mm i.d., dp 1.8 μm) with gradient elution at a flow-rate of 0.3 ml/min. The mobile phase consisted of 10 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring (SRM) mode via electrospray ionization. Target ions were monitored at [M+H]⁺ m/z 498 → 416 and 430 → 372 in positive electrospray ionization (ESI) mode for cymipristone and IS, respectively. Linearity was established for the range of concentrations 0.5–100 ng/ml with a coefficient correlation (r) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.5 ng/ml. The validated method was successfully applied to study the pharmacokinetics of cymipristone in healthy Chinese female subjects.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Enantioselective determination of doxazosin in human plasma by liquid chromatography–tandem mass spectrometry using ovomucoid chiral stationary phase

An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5 mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452 → 344 for doxazosin enantiomers, and m/z 384 → 247 for prazosin (internal standard). The method was linear in the concentration range of 0.100–50.0 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0–11.1% and 5.7–7.6% for R-(−)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4–99.5% for R-(−)-doxazosin and 96.8–102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μM range for GSH and 0.05–20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

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.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

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.     

Simultaneous determination of clarithromycin,rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to ?10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.