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.     

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.     

Simultaneous and sensitive determination of xanthotoxin,psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography–electrospray ionization mass spectrometry

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C₁₈ column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of ?8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

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.     

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.     

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography–electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC™) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 μmol/L (UMP and AMP) to 1.3 μmol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.     

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.     

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.     

Quantification of immature and mature collagen crosslinks by liquid chromatography–electrospray ionization mass spectrometry in connective tissues

We describe a novel high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) method for the simultaneous quantification of enzymatic immature (dihydroxylysinonorleucine DHLNL, hydroxylysinonorleucine HLNL) and mature (pyridinoline PYD, deoxypyridinoline DPD) collagen crosslinks in connective tissues. The crosslinks were separated on a C18 Atlantis^® T3 reversed-phase column with heptafluorobutyric acid (HFBA) as volatile ion-pairing reagent in an acetonitrile–water mobile phase. Detection was carried out by electrospray ionization mass spectrometry in a positive ion mode with selected ion recording (SIR). This method is more sensitive and selective than ion exchange chromatography with post-column ninhydrin detection which is the reference method used for the simultaneous quantification of collagen enzymatic divalent and trivalent crosslinks. The intra and inter-day precision errors were less than 3.4 and 7.7%, respectively for DHLNL, 3.5 and 5.9%, respectively for HLNL, 4.0 and 5.2%, respectively for PYD, 8.2 and 10.7%, respectively for DPD. This novel technique should be useful to quantify simultaneously DHLNL, HLNL, PYD and DPD in connective tissues and to evaluate the maturation of collagen by determination of the ratio between immature and mature enzymatic crosslinks.     

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.     

Simultaneous determination of selegiline-N-oxide,a new indicator for selegiline administration,and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)–electrospray ionization mass spectrometry (HPLC–ESI–MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C₁₈ cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.×150 mm) for HPLC separation and ESI–MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile–10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10–20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users’ urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.     

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 lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

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.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.     

Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography–electrospray ionization tandem mass spectrometry

Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53–7.09% and 0.40–2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells.     

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein–glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [¹³C₂,⁵N]GSH and [¹³C₄,⁵N₂]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.