Liquid chromatography-mass spectrometry method for determination of tetramethylpyrazine and its metabolite in dog plasma

A liquid chromatography-mass spectrometry method is described for the determination of tetramethylpyrazine (TMP) and its active metabolite, 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) in dog plasma. This method involves a plasma clean-up step using protein precipitation procedure followed by LC separation and positive electrospray ionization mass spectrometry detection (ESI-MS). Chromatographic separation of the analytes was achieved on a C18 column using a mobile phase of methanol, water and acetic acid (50:50:0.6, v/v/v) at a flow rate of 1.0 ml/min. Selected ion monitoring (SIM) mode was used for analyte quantitation at m/z 137.2 for TMP, m/z 153.2 for HTMP and m/z 195.2 for caffeine. The linearity was obtained over the concentration ranges of 20-6000 ng/ml for TMP and 20-4000 ng/ml for HTMP and the lower limit of quantitation was 20 ng/ml for both analytes. For each level of QC samples, both inter- and intra-day precisions (R.S.D.) were 相似文献   

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma

A reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra? MS C(18) column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5-2000 ng/ml in plasma for the total drug and 0.5-200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Simultaneous determination of gemcitabine and gemcitabine-squalene by liquid chromatography-tandem mass spectrometry in human plasma

Gemcitabine-squalene is a new prodrug that self-organizes in water forming nanoassemblies. It exhibits better anti-cancer properties in vitro and in vivo than gemcitabine. A liquid chromatography/tandem mass spectrometry assay of gemcitabine-squalene and gemcitabine was developed in human plasma in order to quantitate gemcitabine and its squalene conjugate. After protein precipitation with acetonitrile/methanol (90/10, v/v), the compounds were analyzed by reversed-phase high performance liquid chromatography and detected by tandem mass spectrometry using multiple reaction monitoring. The method was linear over the concentration range of 10-10,000 ng/ml of human plasma for both compounds with an accuracy lower than 10.4% and a precision below 14.8%. The method showed a lower limit of quantitation of 10 ng/ml of human plasma for dFdC and dFdC-SQ. A preliminary in vivo study in mice was shown as application of the method as no significant difference between human and mice plasma for the analysis of dFdC and dFdC-SQ was demonstrated.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Determination of meloxicam in human plasma by liquid chromatography-tandem mass spectrometry following transdermal administration

A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine meloxicam of low concentration in human plasma. After a simple sample preparation procedure by one-step protein precipitation with methanol, meloxicam and the internal standard piroxicam were chromatographed on a Zorbax SB C(18) column. The mobile phase consisted of acetonitrile-water-formic acid (80:20:0.2, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method had a lower limit of quantification of 0.10 ng/ml. The calibration curve was demonstrated to be linear over the concentration range of 0.10-50.0 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were within +/-2.5%), precise (intra- and inter-day relative standard deviation (R.S.D.) <7%). The validated method was successfully applied to the determination of meloxicam in human plasma collected up to 180 h after a transdermal administration of 30 mg meloxicam for evaluation of the pharmacokinetics.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Sensitive determination of vinorelbine and its metabolites in human serum using liquid chromatography-electrospray mass spectrometry

A liquid chromatography-electrospray mass spectrometry method was developed for the quantitation of vinorelbine (VNB) and two metabolites, vinorelbine N-oxide (VNO) and deacetyl vinorelbine (DAV) in human serum. The limits of quantitation (LOQ) reached 0.5 ng/ml for both VNB and VNO and 1 ng/ml for DAV. The method was proved linear in the range of LOQs up to 1000 ng/ml, and extraction recovery was 80% on average for the three compounds. It was applied to the pharmacokinetic monitoring of vinorelbine and, for the first time, to the detection of VNO in the serum of patients suffering from non-small-cell lung cancer.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

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.     

Determination of tamsulosin in dog plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of tamsulosin in dog plasma. Tamsulosin was extracted from plasma using a mixture of hexane-ethyl acetate (2:1, v/v) and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of methanol, water and formic acid (80:20:1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Atmospheric pressure chemical ionization (APCI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 409-->m/z 228 and m/z 256-->m/z 166.9 were used to quantify tamsulosin and the internal standard, respectively. The linearity was obtained over the concentration range of 0.1-50.0 ng/ml for tamsulosin and the lower limit of quantitation was 0.1 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 5.0 and 4.0% (relative standard deviation (R.S.D.)), respectively, and accuracy was within +/-0.3% (relative error (R.E.)). This method was successfully applied to pharmacokinetic study of a tamsulosin formulation product after oral administration to beagle dogs.     

Determination of metolazone in human blood by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of metolazone in human blood. Metolazone was extracted from blood using ethyl acetate and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of acetonitrile, 10 mmol/l ammonium acetate and formic acid (60:40:0.1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Electrospray ionization (ESI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 366-->m/z 259 and m/z 321-->m/z 275 were used to quantify metolazone and the lorazepam (internal standard), respectively. The linearity was obtained over the concentration range of 0.5-500 ng/ml for metolazone and the lower limit of quantitation (LLOQ) was 0.5 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 8.07 and 3.56% (relative standard deviation (RSD)), respectively, and the bias was within +/-4.0%. This method was successfully applied to the pharmacokinetic study of metolazone formulation after oral administration to humans.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

Determination of 25-OH-PPD in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-dammarane-3beta,12beta,20,25-tetrol (25-OH-PPD) in rat. Ginsenoside Rh(2) was employed as an internal standard. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. The mobile phase consisted of methanol-acetonitrile-10 mmol/l aqueous ammonium acetate (42.5:42.5:15, v:v:v), which was pumped at 0.4 ml/min. The analytical column (50 mm x 2.1 mm i.d.) was packed with Venusil XBP C8 material (3.5 microm). The standard curve was linear from 10 to 3000 ng/ml. The assay was specific, accurate (accuracy between -1.19 and 2.57% for all quality control samples), precise and reproducible (within- and between-day precisions measured as relative standard deviation were <5% and <7%, respectively). 25-OH-PPD in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 6h. The method had a lower limit of quantitation of 10 ng/ml, which offered a satisfactory sensitivity for the determination of (25-OH-PPD) in plasma. This quantitation method was successfully applied to pharmacokinetic studies of 25-OH-PPD after both an oral and an intravenous administration to rats and the absolute bioavailability is 64.8+/-14.3%.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

Bioanalysis of tobramycin for therapeutic drug monitoring by solid-phase extraction and capillary zone electrophoresis

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis (CZE) for the analysis of tobramycin in human serum is presented. An off-line SPE employing a carboxypropyl bonded phase (CBA) cartridge was used for the extraction of tobramycin from human serum. Adsorbed tobramycin was eluted from the CBA cartridge using a mixture of NH(3) (25%, w/v)-methanol (30:70, v/v). After evaporation, the analyte was reconstituted and derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The resulting tobramycin-OPA/MPA derivative was purified, and then identified by mass spectrometry. The tobramycin-OPA/MPA derivative was then analysed by CZE with a background electrolyte (BGE) comprising of 30 mM sodium tetraborate pH 10.0-acetonitrile (ACN) (80:20, v/v) with ultraviolet detection at 230 nm. A linear response was observed in the range of 0.3-30 microg/ml with r(2) = 0.992. The sensitivity of the method was determined by its limit of quantitation (LOQ) and limit of detection (LOD) of 0.3 microg/ml and 0.1 microg/ml, respectively. SPE recovery ranged from 68 to 79% at the trough levels to 98% at the peak levels found in serum. Furosemide has been added as internal standard (IS) to improve precision. For the therapeutic range of tobramycin in serum (2-10 microg/ml) the relative standard deviation (R.S.D.) was less than 11% for the entire SPE/CE process. The method demonstrated excellent selectivity as shown by the lack of interference from a total of 20 drugs investigated. The method was then used in therapeutic drug monitoring of patients receiving the drug.     

Liquid chromatography-mass spectrometry method for determination of ramipril and its active metabolite ramiprilat in human plasma

A fast and robust liquid chromatography-mass spectrometry (LC-MS-MS) method has been developed for simultaneous quantitation of the angiotensin-converting enzyme (ACE) inhibitor, ramipril and its metabolite ramiprilat in human plasma. The method involves a solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables a detection limit at sub-nanogram levels. The proposed method has been validated with a linear range of 0.5-250 ng/ml for both ramipril and ramiprilat. The overall recoveries for ramipril and ramiprilat were 88.7 and 101.8%, respectively.