Liquid chromatography tandem mass spectrometry method for simultaneous determination of metoprolol tartrate and ramipril in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol tartrate (MT) and ramipril, in human plasma. Both the drugs were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v). The chromatographic separation was performed on a reversed-phase C8 column with a mobile phase of 10 mM ammonium formate-methanol (3:97, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 5-500 ng/ml for metoprolol and ramipril in human plasma. The precursor to product ion transitions of m/z 268.0-103.10 and m/z 417.20-117.20 were used to measure metoprolol and ramipril, respectively.     

Simultaneous determination of metoprolol succinate and amlodipine besylate in human plasma by liquid chromatography-tandem mass spectrometry method and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by simple liquid-liquid extraction with chloroform. The chromatographic separation was performed on a reversed-phase peerless basic C18 column with a mobile phase of methanol-water containing 0.5% formic acid (8:2, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 1-100ng/ml for MPS and 1-15ng/ml AM in human plasma. The MRM transition of m/z 268.10-103.10, m/z 409.10-334.20 and m/z 296.00-205.10 were used to measure MPS, AM and HCTZ (IS), respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of MPS and AM formulation product after an oral administration to Indian healthy human volunteers.     

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.     

Genome-wide location analysis reveals an important overlap between the targets of the yeast transcriptional regulators Rds2 and Adr1

A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of tetrahydrobiopterin (BH4) and dopamine in rat brain using epsilon-acetamidocaproic acid (AACA) as an internal standard. Proteins in the samples were precipitated with acetonitrile and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 × 100 mm, i.d., 3 μm) column using a mixture of 10mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300 μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) and positive ion mode from m/z 242.1 → 166.0 for BH4, m/z 154.1 → 90.0 for dopamine and m/z 174.1 → 114.0 for AACA (IS). The total chromatographic run time was for 5.5 min. The method was validated for the analysis of samples: the limit of detection was 10 ng/g. The calibration curve was linear between 10-2000 ng/g for BH4 (r(2)=0.995) and 10-5000 ng/g for dopamine (r(2)=0.997) in the rat brain. Thus, good correlated LC-ESI/MS/MS results were obtained and found to be a powerful tool for the quantitative analysis of BH4 and dopamine in the rat brain.     

Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry.

A method for determining the site and extent of deuterium (D) labeling of glucose by GC/MS and mass fragmentography was developed. Under chemical and electron impact ionization, ion clusters m/z 328, 242, 217, 212, and 187 of glucose aldonitrile pentaacetate and m/z 331 and 169 of pentaacetate derivative were produced. From the mass spectra of 13C- and D-labeled reference compounds, glucose carbon and hydrogen (C-H) positions included in these fragments were deduced to be m/z 328 = C1-C6, 2,3,4,5,6,6-H6; m/z 331 = C1-C6, 1,2,3,4,5,6,6-H7; m/z 169 = C1-C6, 1,3,4,5,6,6-H6; m/z 187 = C3-C6, 3,4,5,6,6-H5; m/z 212 = C1-C5, 2,3,4,5-H4; m/z 217 = C4-C6, 4,5,6,6-H4; and m/z 242 = C1-C4, 2,3,4-H3. After correction for isotope discrimination and deuterium-hydrogen exchange, the D enrichment of these fragments can be quantitated using selective ion monitoring, and the D enrichment of all C-H positions can be obtained by the difference in enrichment of the corresponding ion pairs. The validity of this approach was tested by examining D enrichment of known mixtures of 1-d1-, 2-d1-, 3-d1-, and 5,6,6-d3-glucose with unlabeled glucose and D enrichment of perdeuterated glucose using these fragments. This method was used to determine deuterium incorporation in C1 through C6 of blood glucose in fasted (24 h) rats infused with deuterated water. The distribution of deuterium was similar to that found by Postle and Bloxham (1980, Biochem. J. 192, 65-73). Approximately one deuterium atom was incorporated into C5 and only 75% deuterium atom was incorporated into C2. The enrichment of C2 and C6 of glucose relative to that of water indicated that 74 +/- 9% of plasma glucose was newly formed 4 h after the onset of deuterium infusion, and gluconeogenesis accounted for about 76 +/- 7% of the glucose 6-phosphate flux.     

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C(8) micro-bore column (250 mm x 2.1 mm i.d.; 3.5 microm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%.     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, 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 reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of hydroxysafflor yellow A in human plasma: application to a pharmacokinetic study

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in human plasma. HSYA was extracted from human plasma by using solid-phase extraction technique. Puerarin was used as the internal standard. A Shim-pack VP-ODS C(18) (150mm x 4.6mm, 5 microm) column and isocratic elution system composing of methanol and 5mM ammonium acetate (80:20, v/v) provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611.19-->491.19 for HSYA and m/z 415.19-->295.10 for puerarin. The proposed method has been validated with a linear range of 1-1000 ng/ml for HSYA with a correlation coefficient >/=0.999. The lower limit of quantitation was 1 ng/ml. The intra-batch and inter-batch precision and accuracy were within 10%. The average extraction recovery was 81.7%. The total run time was 5.5 min. The validated method was successfully applied to the study on pharmacokinetics of HSYA in 12 healthy volunteers after a single oral administration of safflower oral solution containing 140 mg of HSYA.     

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.     

High-performance liquid chromatography for the determination of 3-n-butylphthalide in rat plasma by tandem quadrupole mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 3-n-butylphthalide in rat plasma. Following protein precipitation with acetonitrile, 3-n-butylphthalide and glipizide (internal standard, I.S.) were separated using a gradient elution program on a C18 column and detected by mass spectrometry in positive ion mode with the multiple reaction monitoring (MRM) mode using the respective precursor to product ion combinations of m/z 191/145 for 3-n-butylphthalide and m/z 446/321 for glipizide, respectively. The total chromatographic running time was 2.5 min. The method was linear over the concentration range of 11.14-3480.00 ng/mL, using as little as 100 microL plasma. The lower limit of quantification (LLOQ) was 5.57 ng/mL. Finally, the method was successfully used to support a preclinical pharmacokinetic study of 3-n-butylphthalide in rats following intravenous administration.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

Rapid and simultaneous determination of tacrolimus (FK506) and diltiazem in human whole blood by liquid chromatography-tandem mass spectrometry: application to a clinical drug-drug interaction study

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI(+)). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7-->768.9 (m/z) for FK506, 415.5-->310.3 (m/z) for DTZ and 809.8-->757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5-200 ng/mL for FK506 and 2-250 ng/mL for DTZ. The recoveries of liquid-liquid extraction method were 58.3-62.6% for FK506 and 50.4-58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.     

Determination of clenbuterol in human urine by GC-MS-MS-MS: confirmation analysis in antidoping control

This work presents a GC-MS-MS-MS method for the direct determination of clenbuterol in human urine. The method comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC-MS(3) (ion trap) with electron impact ionization. The GC-MS(3) analysis allows isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z=335-337) characteristic of clenbuterol. The MS(2) product fragment m/z=300 is in turn used as a further precursor fragment giving rise to a MS(3) spectrum specific for clenbuterol. MS(4) fragmentation spectra were also investigated. However, further fragmentation of MS(3) product ions does not lead to functional MS(4) spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS(3) technique is lower than 0.2 microg/l, with a linear range between 0.5 and 5 microg/l, thus matching the basic requirements for antidoping analysis according to the guidelines of the International Olympic Committee. Due to its overall analytical performance, the method is presently being evaluated as a confirmation protocol to be followed to detect illicit clenbuterol administration to the athletes, and compared with reference GC-MS and GC-MS-MS techniques.     

Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography-tandem mass spectrometry

A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278>262 for DMS and m/z 263>247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax C18 reversed-phase chromatographic column by isocratic elution with 10(-3)M ammonium acetate and 10(-3)M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5-50.0 ng mL(-1)) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94-104% for DMS and 92-106% for the I.S. The intra- and inter-assay precisions were 4.6-8.4% and 2.9-10.6%, respectively. The limit of quantification was 0.15 ng mL(-1). The devised assay was successfully applied to the residual concentrations monitoring in infant.     

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.     

Determination of carbamazepine and carbamazepine 10,11-epoxide in human plasma by tandem liquid chromatography-mass spectrometry with electrospray ionisation

A sensitive method for the determination of carbamazepine and carbamazepine 10,11-epoxide in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated on a Phenomenex Luna C18 5 microm. 150 x 2 mm column with a mobile phase consisting of acetonitrile, methanol and formic acid (0.1%) (10:70:20, v/v). Detection was performed by a Micromass Quattro Ultima mass spectrometer in the MRM mode (LC-MS-MS) using electro spray ionisation (ESI+), monitoring the transition of the protonated molecular ion for carbamazepine at m/z 237.05 and carbamazepine 10,11-epoxide at m/z 253.09 to the predominant ions of m/z 194.09 and 180.04, respectively. The mean recovery was 95% for carbamazepine and 101% for carbamazepine 10,11-epoxide, with a lower limit of quantification of 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine 10,11-epoxide, when using 0.5 ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

Liquid chromatography-electrospray mass spectrometry determination of carbamazepine, oxcarbazepine and eight of their metabolites in human plasma

Carbamazepine (CBZ) and oxcarbazepine (OXCBZ) are both antiepileptic drugs, which are prescribed as first-line drugs for the treatment of partial and generalized tonic-clonic epileptic seizures. In this paper, a specific and sensitive liquid chromatography-electrospray ionization mass spectrometry method was described for the simultaneous determination of carbamazepine (CBZ), oxcarbazepine (OXCBZ) and eight of their metabolites [CBZ-10,11-epoxide (CBZ-EP), 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (DiOH-CBZ), 10-hydroxy-10,11-dihydroCBZ (10-OH-CBZ), 2-hydroxycarbamazepine (2-OH-CBZ), 3-hydroxycarbamazepine (3-OH-CBZ), iminostilbene (IM), acridone (AO) and acridine (AI)] in human plasma. The work-up procedure involved a simple precipitation with acetone. Separation of the analytes was achieved within 50 min using a Zorbax eclipse XD8 C8 analytical column. The mobile phase consisted of a mixture of acetonitrile-formate buffer (2 mM, pH 3). Detection was performed using a quadrupole mass spectrometer fitted with an electrospray ion source. Mass spectrometric data were acquired in single ion recording mode at m/z 237 for CBZ, m/z 180 for CBZ-EP and AI, m/z 236 for OXCBZ, m/z 237 for 10-OH-CBZ, m/z 253 for 2-OH-CBZ, 3-OH-CBZ and DiOH-CBZ, m/z 196 for AO and m/z 194 for IM. For all analytes, the drug/internal standard peak height ratios were linked via a quadratic relationship to plasma concentrations. The extraction recovery averaged 90% for CBZ, 80% for OXCBZ and was 80-105% for the metabolites. The lower limit of quantitation was 0.5mg/l for CBZ, 0.4 mg/l for OXCBZ and ranged from 0.02 to 0.3 mg/l for the metabolites. Precision ranged from 2 to 13% and accuracy was between 86 and 112%. This method was found suitable for the analysis of plasma samples collected during therapeutic drug monitoring of patients treated with CBZ or OXCBZ.     

Quantification of acetaminophen (paracetamol) in human plasma and urine by stable isotope-dilution GC-MS and GC-MS/MS as pentafluorobenzyl ether derivative

We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d(0)) in human plasma and urine by GC-MS and GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d(4)) was used as the internal standard. NAPAP-d(0) and NAPAP-d(4) were extracted from 100-μL aliquots of plasma and urine with 300 μL ethyl acetate (EA) by vortexing (60s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 μL). PFB-Br (10 μL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 μL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 μL of toluene, from which 1-μL aliquots were injected in the splitless mode. GC-MS quantification was performed by selected-ion monitoring ions due to [M-PFB](-) and [M-PFB-H](-), m/z 150 and m/z 149 for NAPAP-d(0) and m/z 154 and m/z 153 for NAPAP-d(4), respectively. GC-MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 → m/z 107 and m/z 149 → m/z 134 for NAPAP-d(0) and m/z 154 → m/z 111 and m/z 153 → m/z 138 for NAPAP-d(4). The method was validated for human plasma (range, 0-130 μM NAPAP-d(0)) and urine (range, 0-1300 μM NAPAP-d(0)). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r>0.999) was found between the concentrations measured by GC-MS and GC-MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 μL). The analytical performance of the method makes it especially useful in pediatrics.     

A sensitive and specific HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of isoforskolin in beagle dogs

A sensitive and specific high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. Liquid-liquid extraction was used to extract isoforskolin and the internal standard (I.S.) eplerenone from canine plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol-2mM ammonium acetate-formic acid (62:38:0.1, v/v/v), pumped at 0.35 mL/min. Isoforskolin and I.S. were detected at m/z 433.4→373.3 and m/z 415.3→163.5 in positive ion and multiple reaction monitoring (MRM) mode, respectively. The standard curves were linear over the concentration range of 0.1-200 ng/mL (r>0.99). The intra- and inter-batch accuracy values for isoforskolin at four concentrations were 90.2-108.3% and 97.8-106.6%, respectively. The RSDs were less than 6.0%. The mean extraction recoveries of isoforskolin and I.S. were 97.0 and 88.4%, respectively. The method was successfully applied to the pharmacokinetic study after an intravenous administration of isoforskolin in beagle dogs.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.