Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.     

Determination of phloroglucinol in human plasma by high-performance liquid chromatography-mass spectrometry

A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.     

Determination of rimonabant in human plasma and hair by liquid chromatography-mass spectrometry

Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50muL of plasma or 10mg of hair using diethylether. Chromatography was performed on a 150mmx2.1mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/z 463.1 for rimonabant and m/z 555.1 for IS. The most intense product ion of rimonabant (m/z 380.9) and IS (m/z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85-115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at -20 degrees C. This simple and robust LC-MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.     

Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [¹⁵N,¹³C₂]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine⁻¹ were found in healthy controls, with reduced levels in hemochromatosis (P < 0.00006) and elevated levels in inflammation (P < 0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P < 0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.     

Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography-mass spectrometry

A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography-mass spectrometry (LC-MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC-MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were 相似文献   

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.     

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.     

Determination of triethylenetetramine (TETA) and its metabolites in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS)

A liquid chromatography-mass spectrometry (LC-MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC-MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 M Omega), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M+H]+ values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.     

Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction

A high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were < or = 40 nM for the parent compounds and < or = 25 nM for the metabolites and the limit of detection (LOD) was < or = 2.5 nM for all analytes. Average recovery was 84% (+/- 3.7) and 74% (+/- 13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were < or = 8.6 and < or = 10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics.     

Determination of carboplatin in plasma and tumor by high-performance liquid chromatography-mass spectrometry

Carboplatin is a platinum analogue that is used in a number of chemotherapeutic regimens for solid tumors, such as lung and ovarian carcinomas. Most often characterization of carboplatin's pharmacokinetic properties is based on measurement of platinum, rather than intact carboplatin. We have developed a sensitive LC-MS method for the determination of intact carboplatin in plasma ultrafiltrate and in tumor tissue. Carboplatin was extracted from rat plasma ultrafiltrate and tumor samples using solid-phase extraction cartridges and analyzed using reversed-phase chromatography with positive electrospray ionization followed by mass spectrometric detection. Using 50 microliter of plasma ultrafiltrate or 140 microliter of tumor homogenate supernatant, the extraction afforded a recovery of 58.7 and 45.8% for plasma and tumor, respectively. The mobile phase was 5% acetonitrile in 0.5% acetic acid at 0.2 ml/min that yielded a retention time of carboplatin of 2.2 min. The method has been validated at carboplatin plasma ultrafiltrate concentrations from 0.07 to 2.5 microgram/ml, and from 0.03 to 1.3 microgram/ml in tumor homogenates. The main advantages of this method compared with earlier methods are the ability to measure intact carboplatin in a sensitive and specific manner.     

Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography-mass spectrometry

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of (R)- and (S)-ketamine, and (R)- and (S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC-MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized alpha(1)-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol-ammonium acetate buffer (10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 degrees C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.     

Determination of L-threonate in human plasma and urine by high performance liquid chromatography-tandem mass spectrometry

A fast and selective HPLC-MS-MS method was established to determine L-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC J'Sphere C(18) column with methanol-acetonitrile-10mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and L-threonate was quantified by monitoring the ion transition of m/z 134.5-->74.7. The linear calibration curves of L-threonate in plasma and urine were obtained over the concentration range of 0.25-50 microg/ml and 2.5-500 microg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 microg/ml, respectively. Accuracy was within 85-115%, and intra- and inter-batch precision (R.S.D.%) were within +/-15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of L-threonate in Chinese healthy subjects.     

Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography-mass spectrometry

Paclitaxel is an anticancer agent extracted from the bark of the yew tree and is widely used in chemotherapy for solid tumors, including non-small cell lung cancer and ovarian carcinoma. Most assays to measure paclitaxel in plasma require a large amount of sample (0.4-1 ml) to achieve the necessary sensitivity, and are not suitable when only small sample sizes are available. To circumvent this latter limitation, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) method for the determination of paclitaxel in plasma based on the use of small sample volumes (50 microl plasma). A solid phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of paclitaxel were 98 and 83% from plasma and brain tissues, respectively. The mobile phase consisted of 50% acetonitrile in 0.1% formic acid that was pumped at 0.2 ml/min to yield a retention time for paclitaxel of 6.2 and 5.4 min for cephalomannine, the internal standard. The method has been validated at paclitaxel plasma concentrations from 0.036 to 9.9 microg/ml, and from 0.054 to 1.96 microg/ml in brain homogenates. A sensitive and specific assay for paclitaxel has been developed that has the advantages of using small sample sizes, and a single extraction step without solvent evaporation.     

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry

24S-Hydroxycholesterol (24S-OH-Chol) and 27-hydroxycholesterol (27-OH-Chol) are oxidized derivatives of cholesterol and of potential diagnostic interest because their circulating levels may reflect the cholesterol metabolism of the brain and macrophages, respectively. We developed a sensitive and specific HPLC-MS method for the quantification of 24S-OH-Chol and 27-OH-Chol in human plasma. In contrast to currently available procedures based on gas chromatography-mass spectrometry, this methodology offers the advantage that no time-consuming derivatization is needed. After saponification, solid-phase extraction, and HPLC separation under reversed-phase column conditions, detection by MS was performed using atmospheric pressure chemical ionization and selected ion monitoring mode. The standard curves were linear throughout the calibration range for both oxysterols. Within-day and between-day coefficients of variation were less than 9%, and the recoveries ranged between 98% and 103%. The quantification limits were 40 and 25 microg/l for 24S-OH-Chol and 27-OH-Chol, respectively. Mean values for both oxysterols were determined in plasma from 22 healthy volunteers. The sensitive and selective HPLC-MS method described here combined with the appropriate workup procedure allow the quantification of 24S-OH-Chol and 27-OH-Chol in plasma samples, for example in clinical studies to elaborate the clinical usefulness of these two oxysterols.