Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

Determination of Memantine in Human Plasma by LC–MS–MS: Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of memantine was developed and validated over the linearity range 0.1–25 ng mL^?1 with 0.5 mL of plasma using procainamide as the internal standard. This analysis was carried out on a Cosmosil 5C₁₈-MS column and the mobile phase was composed of methanol: 0.5% formic acid (50:50, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization and quantification was performed by multiple reaction monitoring mode. The MS–MS ion transitions monitored were m/z 180 → 107 and 236 → 163 for memantine and procainamide, respectively. The between- and within-day precision was less than 10.9% and accuracy was less than 2.5%. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of memantine in healthy Chinese volunteers.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

Determination of Doxapram Hydrochloride in Rabbit Plasma by LC-MS-MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of doxapram hydrochloride in rabbit plasma was developed. After addition of urapidil hydrochloride as internal standard (IS), protein precipitation by 10% trichloroacetic acid in methanol (w/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 50 mm, 3.5 μm) column with acetonitrile–water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 378.9 → 291.8 for doxapram hydrochloride and m/z 387.9 → 204.6 for the IS. Calibration plots were linear over the range of 2–1000 ng mL^?1 for doxapram hydrochloride in plasma. Lower limit of quantitation (LLOQ) for doxapram hydrochloride was 2 ng mL^?1. Mean recovery of doxapram hydrochloride from plasma was in the range 83.7–91.5%. RSD of intra-day and inter-day precision were less than 9%, respectively. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of doxapram hydrochloride in rabbit plasma.     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Simultaneous Determination of Buprenorphine, Norbuprenorphine and Naloxone in Human Plasma by LC-MS-MS

A novel sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method simultaneously determined buprenorphine (BUP) and its active metabolite, norbuprenorphine (NBUP), and a coformulant, naloxone was developed, validated and applied successfully in humans. Buprenorphine-d ₄ and norbuprenorphine-d ₃ were used as the internal standard. The analysis was performed on a silica column, and the mobile phase was isocratic and composed of acetonitrile:2 mM ammonium formate in H2O (82:18, v/v). Mass spectrometry employed multiple reaction monitoring modes with transitions of m/z 468.1?C55.2 for BUP, 414.2?C101.2 for NBUP, 328.3?C310.3 for naloxone, 472.1?C59.2 for buprenorphine-d ₄ and 417.2?C101.2 for norbuprenorphine-d ₃. Lower limit of quantification (LLOQ) of the analytical method was 0.05 ng mL^?1 for BUP, 0.1 ng mL^?1 for NBUP and 0.025 ng mL^?1 for naloxone, respectively. The standard calibration curves of BUP, NBUP and naloxone were linear over the concentration range of 0.05?C20 ng mL^?1, 0.1?C20 ng mL^?1 and 0.025?C20 ng mL^?1, respectively. The precisions (RSD) and accuracies (RE) of LLOQ and other QC samples were in acceptable range, with RSD < 20% and RE ± 20% for LLOQ and RSD < 15% and RE within ±15% for QC samples. The method was accurate, precise and specific, and was applied to the pharmacokinetic study of buprenorphine in healthy volunteers.     

Simultaneous Determination of Pregabalin, Sildenafil and Its Active Metabolite in Rat Plasma Utilising SPE Followed by LC�CMS�CMS

A sensitive and selective analytical method for the quantification of pregabalin, sildenafil and the active desmethyl metabolite of sildenafil (UK-103320) has been developed. The method can simultaneously quantify the three analytes within the expected in vivo concentration ranges using 50 ??L of rat plasma. It utilises solid-phase extraction followed by high performance liquid chromatography coupled with tandem mass spectrometry. Quantitation in rat plasma demonstrated good accuracy and precision over the following dynamic ranges for each analyte: pregabalin (70?C10,000 ng mL^?1), sildenafil (1?C2,000 ng mL^?1) and UK-103320 (1?C2,000 ng mL^?1). For each analyte, the following lower limits of quantitation were obtained: 70 ng mL^?1 for pregabalin and 1 ng mL^?1 for sildenafil and UK-103320, respectively. The method was successfully used to analyse plasma samples from rats when pregabalin and sildenafil were administered in combination.     

LC-MS-MS Determination and Pharmacokinetic Study of Clozapine in Human Plasma

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry was established and validated for the determination and pharmacokinetic study of clozapine in human plasma. Ethyl acetate extraction was used for plasma sample preparation with mirtazapine as internal standard. Chromatographic separation was achieved on a Hanbon Kromasil C₁₈ (250 mm × 4.6 mm, 5 μm) column by isocratic elution with a mixture of 70 volumes of methanol and 30 volumes of water containing 0.2% ammonium acetate and 0.1% formic acid as mobile phase delivered at 1.0 mL min^?1. The MS-MS detection was carried out on a tandem mass spectrometer using positive electrospray ionization and multiple reaction monitoring with argon for collision-induced dissociation. The ion transitions were monitored as follows: m/z 327 to m/z 270 for clozapine and m/z 266 to m/z 195 for the internal standard (mirtazapine), respectively. Calibration curves were generated over the concentration range from 0.10 to 200 ng mL^?1 with the lower limit of quantification of 0.10 ng mL^?1, and two segments of linear calibration curves were established by regressing in the way of least-square in the range from 0.10 to 5.0 and 5.0 to 200 ng mL^?1, respectively. The intra- and inter-day precision and accuracy were determined at three different concentration levels, 0.20, 10.0 and 100 ng mL^?1, and were all better than 15% (n = 5). This specific and sensitive liquid chromatography coupled with tandem mass spectrometry has been successfully applied to a pharmacokinetic study of clozapine after a single oral dose of 25 mg in healthy Chinese volunteers.     

Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C₈ reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min^?1. There is good linearity in the range of 0.5–500 ng mL^?1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL^?1 and 0.08 ng mL^?1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL^?1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL^?1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.     

Ion Chromatography in Combination with ESI Ion Trap MS for the Analysis of Diphacinone in Animal Livers

Analysis of diphacinone is often performed on C18 column by reserved phase liquid chromatographic methods coupled with ultraviolet detection or by ion-pair chromatography coupled with mass spectrometry. In this work, a sensitive and selective method using eluent generator reagent free ion chromatography coupled with electrospray ionization ion trap mass spectrometric detection for the quantification of diphacinone in animal livers has been developed. Samples were extracted with 10% (v/v) methanol in acetonitrile after spiking with warfarin (internal standard, IS) and cleaned by solid-phase extraction. The analytes were separated on an IonPac AS11 analytical column (250 mm × 4.0 mm) using 30.0 mmol L^?1 KOH (achieved by a KOH eluent generator) containing 10% (v/v) methanol as organic modifier at a flow rate of 1.0 mL min^?1. Detection was performed by negative electrospray ionization and ion trap tandem mass spectrometry in multiple reaction monitoring mode with a transition of m/z 339 → 167 for diphacinone, and 307 → 161 for IS, respectively. The calibration curve was linear (r ² = 0.993) in the concentration range of 1.0–200.0 ng mL^?1 with a lower limit of quantification of 0.3 ng g^?1 in animal livers. Intra- and inter-day relative standard deviations (RSDs) were less than 8.7 and 12.5%, respectively. Recoveries of diphacinone ranged from 81.2 to 89.5%. It was confirmed that this method could be used in clinical diagnosis or forensic toxicology.     

Determination of Gastrodin and Ligustrazine Hydrochloride in Plasma and Brain Dialysate by LC–Tandem MS

A gradient liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of gastrodin and ligustrazine hydrochloride in rat plasma and brain dialysates. Zolpidem was used as internal standard. For plasma samples, solid-phase extraction was used and the brain dialysates were collected from freely moving rats using brain microdialysis. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI–MS–MS). Chromatographic separation was achieved on a Symmetry RP-18 column using gradient elution with methanol and water containing 0.5% formic acid and 2 mM ammonium formate. Selected reaction monitoring (SRM) mode was used for quantitation. Good linearities were obtained in the range of 0.05–100 and 0.01–50 μg mL^?1 for gastrodin and ligustrazine hydrochloride in rat plasma, and 0.05–1,000 ng mL^?1 for both in dialysate. The lower limit of quantitation was 0.01 ng mL^?1 for gastrodin and 0.05 ng mL^?1 for ligustrazine. The method is precise and reliable and can be applied to pharmacokinetic studies.     

Ocular Pharmacokinetic Study of l-Carnosine and N-Acetyl-l-carnosine in Rabbit by Microdialysis Coupled with UPLC-MS-MS

In order to provide useful information for rational drug design, the ocular pharmacokinetics of l-carnosine (CAR) and its acetylized prodrug N-acetyl-l-carnosine (NAC) were investigated. The in vivo microdialysis sampling coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was developed for continuously simultaneous monitoring of CAR and NAC in rabbit aqueous humor. The measured in vitro recoveries of the probe were 61.3% for CAR and 65.8% for NAC, while in vivo recoveries decreased to 43.1% for CAR and 43.0% for NAC, respectively. The method was sensitive with LLOQ 20.5 ng mL^?1 for CAR and 20.4 ng mL^?1 for NAC. The initial data indicated that the value of C _max and AUC_(0?C??) of NAC were higher than these of CAR (C _max 2305 vs. 1,802 ng mL^?1), (AUC_(0?C??) 1,337 vs. 1,891 ng h mL^?1), which indicated that the NAC exhibited better ocular bioavailability and duration. The method was rapid, specific and sensitive for continuously monitoring of aqueous humor and it was successfully applied to pharmacokinetic studies of CAR and NAC.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

LC�CMS�CMS Method for Quantification of Hydralazine in BALB/C Mouse Plasma and Brain: Application to Pharmacokinetic Study

A rapid, sensitive and accurate high performance liquid chromatography method using tandem mass spectrometry detection for hydralazine in BALB/C mouse plasma and brain was developed and validated. The method involved a derivatization with 2,4-pentanedione at 50 °C for 1 h, and a step of solid phase extraction to purify and concentrate hydralazine derivative. Chromatographic separation was carried out on an Agilent ZORBAX SB-C18 column by elution with methanol?C0.01 mol L^?1 ammonium acetate (60:40, v/v). The multiple reaction monitoring transition used for quantification was m/z 225.2 ?? 129.5 in the electrospray positive ionization mode. Good linearity was obtained over the concentration range of 10?C200 ng mL^?1. The limits of detection were 0.49 and 1.05 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. The limits of quantitation were 1.5 and 3.18 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. Sample analysis time was 6 min including sample separation. The method was successfully applied to a pharmacokinetic study following intraperitoneal injection of hydralazine in BALB/C mice at the dose of 20 mg kg^?1.     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma, and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min^?1. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL^?1 for wogonin and 10.8–271 ng mL^?1 for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL^?1 for wogonin and 10.8 ng mL^?1 for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.     

Determination of 5-Hydroxytryptamine, Norepinephrine, Dopamine and Their Metabolites in Rat Brain Tissue by LC–ESI–MS–MS

A liquid chromatography–electrospray ionization tandem mass spectrometry method has been developed to perform the determination of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites, i.e., 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat brain tissue. Analytes were separated on a Thermo C₁₈ column (4.6 mm × 250 mm, 5 μm, SN: 1245575T, Thermo electron corporation, USA) with a mobile phase of 0.05% formic acid/acetonitrile (92:8 for ESI⁺, 82:18 for ESI^?, v/v) at the flow-rate of 0.8 mL min^?1. The LC system was coupled to a Waters Micromass Quattro Premier XE tandem quadruple mass spectrometer. MS acquisition of 5-HT, NE and DA was performed in positive electrospray ionization multiple reaction monitoring (MRM) mode, while negative electrospray ionization MRM mode was used to monitor their metabolites. The calibration curves were linear within the concentration range of 4–4,450 ng mL^?1 for 5-HT, 4–4,110 ng mL^?1 for NE and 4–4,100 ng mL^?1 for DA (r ≥ 0.999). The limit of quantitation was 4 ng mL^?1. 5-HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12–1,000 ng mL^?1(r ≥ 0.998) and the limit of quantitation was 12 ng mL^?1. The intra- and inter-day RSD were lower than 8.45%. The method is sensitive, fast, accurate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.     

LC–APCI–MS–MS for the Determination of Celastrol in Human Whole Blood

A rapid, sensitive high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) assay has been developed and validated for the quantitative determination of celastrol in human whole blood using hydrocortisone as an internal standard (I.S.). The celastrol and I.S. were extracted from human whole blood with ethyl acetate. The separation was performed by reversed-phase HPLC using an isocratic mobile phase consisting of 5 mmol L^?1 aqueous ammonium acetate containing 0.05% acetic acid/methanol (25:75, v/v) on a XDB-C₁₈ column (150 mm × 4.6 mm I.D., 5 μm). Detection was by negative atmospheric pressure chemical ionization (APCI) and ion trap tandem mass spectrometry in multiple reaction monitoring (MRM) mode with a transition of m/z 449.4 → 405.1 for celastrol, and 419.2 → 329.1 for I.S. The calibration curve was linear (r ² = 0.9967) in the concentration range of 1.0–200.0 ng mL^?1 in human whole blood with a limit of quantification of 1.0 ng mL^?1. Intra-day and inter-day relative standard deviations (RSDs) were less than 8.5 and 10.1%. The mean extraction recovery was 89.2% for celastrol and 92.6% for I.S. This assay can be used to determine trace celastrol in human whole blood.     

The determination of salivary oxypurines before and after exercise by combined liquid chromatography-field asymmetric waveform ion mobility spectrometry-time-of-flight mass spectrometry

A method combining field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of the oxypurine compounds hypoxanthine (HX) and xanthine (XA) in saliva. Separation of the oxypurines from interfering matrix components was investigated using FAIMS-MS. The selected FAIMS parameters were then applied to the rapid LC-FAIMS-MS analysis of HX and XA using a short chromatographic separation method (7 min). A comparison of the LC-MS method with and without FAIMS applied, resulted in improved discrimination from saliva matrix interferences and improved chromatographic peak integration for both HX and XA using a FAIMS separation. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection of 2.0 ng mL^?1 for HX and 1.8 ng mL^?1 for XA, and limits of quantification of 6.6 ng mL^?1 for HX and 6.0 ng mL^?1 for XA. The developed LC-FAIMS-MS method was applied to the targeted analysis of the oxypurine metabolites in saliva collected from healthy male athletes (n =?11) before and after exercise designed to induce oxidative stress; post-exercise collection time-points included immediately after exercise, one hour and twenty-four hours’ post-exercise. The salivary concentrations of both HX and XA were lower after physical exercise, compared to the pre-exercise (rest) concentrations and returned to approximately pre-exercise levels after twenty-four hours. The method reported has the potential for monitoring the salivary oxypurines, HX and XA, as biomarkers of oxidative stress and in other clinical applications.     

Quantification of Tacrolimus in Human Skin Samples and Ointment by LC-MS

A sensitive and simple liquid chromatography-mass spectrometry method was developed to determine the immunosuppressant tacrolimus in human skin samples after treatment with the commercially available ointment. Utilizing diffusion cell experiments according to Franz, human skin samples were treated with ointment containing tacrolimus and the extraction procedure of the drug was optimized. The analytical assay was performed using an LC system consisting of a reversed phase C₁₈ column and an isocratic mobile phase, coupled with electrospray ionization mass spectrometry. The detection was performed in the positive selected ion monitoring mode. Mycophenolate mofetil was used as internal standard to control the stability of the electrospray ionization. The calibration curve was linear for tacrolimus over the range of 5–1,000 ng mL^?1 (average correlation coefficient of r ² = 0.9941) with a limit of detection (LOD) of 2 ng mL^?1, with a limit of quantification (LOQ) of 5 ng mL^?1 and with a precision of 8.70%. The analytical assay described in this paper was successfully applied in order to quantify tacrolimus in human skin samples as well as in the commercially available ointment.