Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.     

Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of alpha-methyltyrosine in human plasma

A sensitive and selective LC-MS-MS method for the isolation and quantification of alpha-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC-MS-MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C(18) column (2.0 mm x 50 mm; 5 microm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water-acetic acid (100:0.1, v/v) and acetonitrile-acetic acid (100:0.1, v/v). The structural analog alpha-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.     

Development and validation of a liquid chromatography-tandem mass spectrometry assay for determination of raloxifene and its metabolites in human plasma

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4'-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC-MS-MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r(2)>0.99) were found from 0.200 to 340 microg/L, from 1.600 to 2720 microg/L and from 0.088 to 60.00 microg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).     

Development and validation of liquid chromatography-tandem mass spectrometric method for simultaneous determination of fosinopril and its active metabolite fosinoprilat in human plasma

A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of the prodrug fosinopril and its major active metabolite fosinoprilat for pharmacokinetic studies in healthy subjects. In order to get the lower limit of quantification (LLOQ), especially for analysis of fosinopril, key points of the method have been investigated including chromatographic conditions and selection of LC-MS/MS conditions. The analytes were extracted from plasma samples by liquid-liquid extraction, separated on a reversed-phase C(8) column using gradient procedure, and detected by tandem mass spectrometry with a triple quadrupole ionization interface. The analytes and internal standard zaleplon were detected using positive electrospray ionization (ESI) in the SRM mode. The LLOQ of the method down to 0.1 ng mL(-1) for fosinopril and 1.0 ng mL(-1) for fosinoprilat were identifiable and reproducible. The standard calibration curves for both fosinopril and fosinoprilat were linear over the ranges of 0.1-15.0 and 1.0-700 ng mL(-1) in human plasma, respectively. The within- and between-batch precisions (relative standard deviation (RSD)%) and the accuracy were acceptable. The validated method was successfully applied to reveal the pharmacokinetic properties of fosinopril and fosinoprilat after oral administration.     

Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of L-homoarginine in human plasma

Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of L-arginine catalysed by NO synthase (NOS). The L-arginine derivative L-homoarginine serves as an alternative NOS substrate releasing NO, competing with L-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography-tandem mass spectrometric (LC-tandem MS) method for the determination of L-homoarginine in human plasma by stable-isotope dilution. L-[(13)C(6)]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC-tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for L-homoarginine and L-[(13)C(6)]-homoarginine were m/z 245 → m/z 211 and m/z 251 → m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1-50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of L-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans.     

Development and validation of a stereoselective liquid chromatography-tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma

A stereoselective liquid chromatography-tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5-50 microg/L in human plasma. Metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range 0.5-50 microg/L in plasma, with 0.5 microg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92-105%.     

Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma

As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.     

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.     

Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of the major metabolites of duloxetine in human plasma

A sensitive bioanalytical method for the measurement of two major circulating metabolites of duloxetine [4-hydroxy duloxetine glucuronide (LY550408) and 5-hydroxy-6-methoxy duloxetine sulfate (LY581920)] in plasma is reported. This method produced acceptable precision and accuracy over the validation range of 1-1000 ng/mL. Several issues had to be addressed in order to develop an LC/MS/MS assay for these metabolites. First, 4-hydroxy duloxetine glucuronide required chromatographic resolution from the 5-, and 6-hydroxy duloxetine glucuronide isomers. Second, the glucuronide conjugate is readily ionized under positive ESI conditions, while the sulfate conjugate required negative ESI conditions to obtain adequate sensitivity. Finally, the chromatographic conditions needed to separate the glucuronide isomers were not suitable for the analysis of the sulfate conjugate. The present method addressed these challenges, and was successfully applied to multiple human pharmacokinetic studies in which subjects received oral doses of duloxetine hydrochloride.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Development and validation of a high performance liquid chromatography-tandem mass spectrometry for the determination of etodolac in human plasma

A simple and specific method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C(18) column with 65% acetonitrile and 35% water containing 10mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99>172.23 for etodolac and m/z 357.92>139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1microg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of galantamine in human heparinised plasma

Galantamine is an acetylcholinesterase inhibitor, recently approved for the treatment of mild-to-moderate Alzheimer's disease. To allow a higher throughput of samples, a new bioanalytical method for the determination of galantamine in human plasma was developed and validated. A stable isotope labelled internal standard was used. Sample preparation consisted of a simple one-step liquid-liquid extraction with toluene. The extracts were analysed with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS). The method was validated in the 1-500-ng/ml range. The accuracy, precision, selectivity, lower limit of quantification, upper limit of quantification, linearity and extraction recovery were evaluated, as well as the stability of the compound in plasma, blood, methanol and 2% BSA solutions under different conditions. The method proved very rugged during the analysis of large numbers of samples from clinical trials.     

Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for dihydroergotamine and its major metabolite in human plasma

A sensitive and specific procedure for the simultaneous determination of dihydroergotamine (DHE) and its 8'-hydroxylated metabolite (8'-OH-DHE) in human plasma was developed and validated. The analytes were extracted from plasma samples by liquid-liquid extraction, separated through a Zorbax C18 column (50x2.1 mm I.D.) and detected by tandem mass spectrometry with an electrospray ionization interface. Caroverine was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 and 11.0 pg/ml for DHE and 8'-OH-DHE, respectively. The intra- and inter-run precision was measured to be below 9.1% for both DHE and 8'-OH-DHE. The inter-run accuracy was within 4% for the analytes. The overall extraction recoveries of DHE and 8'-OH-DHE were determined to be about 58 and 52% on average, respectively. The chromatographic run time was approximately 2.5 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of DHE and 8'-OH-DHE in a pharmacokinetic study of volunteers who received DHE orally.     

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.     

Development and validation of a liquid chromatographic–tandem mass spectrometric method for the determination of pibutidine in human urine

A liquid chromatographic–tandem mass spectrometric method for the rapid quantitative determination of pibutidine, an H₂-receptor antagonist, in human urine has been developed and validated over the concentration range 0.1–25.6 μg ml⁻¹. Urine samples were prepared based on a simple dilution with 0.05% acetic acid, followed by reversed-phase liquid chromatographic separation. Pibutidine and its internal standard (²H₁₀-pibutidine) were ionized using an electrospray ionization interface and detected by tandem mass spectrometry in the selected reaction-monitoring mode. Completed validation demonstrated the method to be robust, accurate, precise and specific for the direct quantification of pibutidine in human urine. This method has enabled investigation of the urinary excretion of pibutidine following oral administration of pibutidine hydrochloride to healthy subjects.     

Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of metformin in human plasma using phenformin as internal standard has been developed and validated. Sample preparation of plasma involved acidification with acetic acid, deproteination with acetonitrile and washing with dichloromethane. Samples were then analyzed by HPLC on a short Nucleosil C18 column (5 microm, 50 mm x 4.6 mm i.d.) using a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate pH 7.0 (20:20:60, v/v/v) delivered at 0.65 ml/min. Detection was performed using an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring (MRM) mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The assay was linear over the range 1-2000 ng/ml with intra- and inter-day precision of <8.6% and accuracy in the range 91-110%. The limit of detection was 250 pg/ml in plasma. The method was successfully applied to a clinical pharmacokinetic study of an extended-release tablet of metformin hydrochloride (500 mg) administered as a single oral dose.     

Development and validation of a sensitive liquid chromatographic-tandem mass spectrometric method for the determination of cromolyn sodium in human plasma

Cromolyn sodium is a safe compound with potent anti-allergic properties when used locally or topically. Clinical data from systemic exposure is not available because of the poor GI absorption when given orally. In order to evaluate a new approach to enhance the absorption and bioavailability of cromolyn sodium, a sensitive assay was needed to support an oral-dose study in humans. This paper describes a liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method for the analysis of cromolyn sodium in human plasma. The method consists of a two-step extraction with subsequent analysis using a high-performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C(18) column followed by a backflush. The total run time is 6 min. The standard curve of cromolyn sodium was over the range of 0.313 to 750 ng/mL with a lower limit of quantitation (LLOQ) of 0.313 ng/mL when 0.5 mL of plasma was used for analysis. The percent coefficient of variation (C.V.) for accuracy and precision (inter-assay and intra-assay) was less than 15% over the validated concentration range and the coefficients of determination, r(2), were >0.991577. The method is simple, sensitive, and selective, and has been successfully utilized for oral cromolyn sodium clinical studies.     

Selective and rapid liquid chromatography-tandem mass spectrometry assay of dutasteride in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of dutasteride (I), a potent and the first specific dual inhibitor of 5alpha-reductase, in human plasma. The analyte and internal standard (finasteride (II)) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Xterra MS C18 column with a mobile phase of 10 mM ammonium formate/acetonitrile (15/85, v/v, pH adjusted to 3.0 with formic acid). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 529.5 --> 461.5 and m/z 373.3 --> 317.4 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1-25.0 ng/mL for dutasteride in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples/day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.