Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl plasma for atazanavir and 50 microl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50 mm x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05-10 microg/ml for atazanavir and 0.1-75 microg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within +/-7.3 and +/-7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC-MS/MS assay for the quantification of protease inhibitors.     

Quantification of tipranavir in human plasma by high-performance liquid chromatography with UV detection

A simple method for the quantification of tipranavir, a new non-peptidic protease-inhibitor, was developed. An internal standard, prazepam, was added to 100 microl of plasma before a liquid-liquid extraction by 3 ml of tert-butyl methyl ether. The extracts were evaporated to dryness and reconstituted with 100 microl of mobile phase before being injected in the chromatographic system. The separation was made on a C8 column using sodium acetate buffer (pH 5):methanol:acetonitrile (35:30:35, v/v/v) as mobile phase. The detection was performed at a wavelength of 260 nm. The method was linear and has been validated over a concentration range of 2-80 mg/l. The mean precision and accuracy of the method were respectively, 10.5 and -9.1%. The mean recovery was 70.8%.     

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.     

Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study

A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm x 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm x 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1-20 microg/ml. With UV detection, the limit of quantitation was 0.1 microg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 microl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 +/- 3.9, 93.5 +/- 3.2, and 93.7 +/- 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 microl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.     

Determination of imatinib (Gleevec) in human plasma by solid-phase extraction-liquid chromatography-ultraviolet absorbance detection

A sensitive HPLC method has been developed for the assay of imatinib in human plasma, by off-line solid-phase extraction followed by HPLC coupled with UV-Diode Array Detection. Plasma (750 microl), with clozapine added as internal standard, is diluted 3 + 1 with water and subjected to a solid-phase extraction on a C18 cartridge. After matrix components elimination with 2000 microl of water (in two aliquots of 1000 microl), imatinib is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 180 microl 50% methanol. A 50 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. Imatinib is analyzed using a gradient elution program with solvent mixture constituted of methanol and water containing both 0.05% ammonium acetate. Imatinib is detected by UV at 261 nm. The calibration curves are linear between 0.1 and 10 microg/ml. The limit of quantification and detection are 0.05 and 0.01 microg/ml, respectively. The mean absolute recovery of imatinib is 96%. The method is precise with mean inter-day CVs within 1.1-2.4%, and accurate (range of inter-day deviations -0.6 to +0.7%). The method has been validated and is currently being applied in a clinical study assessing the imatinib plasma concentration variability in a population of chronic myeloid leukemia- and gastro-intestinal stromal tumor-patients.     

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

Liquid chromatographic determination of isoniazid, pyrazinamide and rifampicin from pharmaceutical preparations and blood

Isoniazid (IN), pyrazinamide (Pz) and rifampicin (Rf) are separated on YMC-ODS column. IN was derivatized with 2-fluorene-carboxaldehyde (FA). The separation was achieved using ethanol-chloroform-acetonitrile water by isocratic elution and detected at 337 nm. The detection limits were 0.11 ng, 0.2 ng and 13 ng/injection (5 microl) for IN, Pz and Rf, respectively. The method of analysis was applied to the pharmaceutical preparations and in the blood samples of the patients suffering from tuberculosis after undergoing chemotherapy with IN, Pz and Rf. The amounts quantitated in blood showed 0.97 to 1.58 microg/ml IN, 3.44 to 4.09 microg/ml Pz and 1.98 to 3.5 microg/ml Rf with coefficient of variations 0.8-1.8%, 0.9-1.3% and 0.8-2.1%, respectively.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

Effects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve.

Alpinia zerumbet, known popularly as "col?nia" in Northeastern Brazil, is a medicinal plant that has been used widely in folk medicine as teas and infusions for the treatment of intestinal and cardiovascular diseases, including arterial hypertension. Our previous studies have demonstrated that the essential oil of A. zerumbet (OEAZ) is very active on excitable tissues, such as smooth muscle, and in this study we verified its effects on the compound action potential (CAP) of rat sciatic nerve. EOAZ induced a dose-dependent blockade of the CAP. Control peak-to-peak amplitude and conduction velocity of CAPs were 7.6 +/- 0.43 mV and 80.6 +/- 3.19 m/s, respectively. At 60 microg/ml, EOAZ induced no demonstrable effect. Conduction velocity was significantly reduced at 180 min of preparation exposure to 100 microg/ml of EOAZ. At 300, 600 and 2000 microg/ml doses of EOAZ, the peak-to-peak amplitudes of CAPs following 180 min exposure of the nerve to the drug were reduced significantly, to 75.3 +/- 7.36%, 50.45 +/- 2.17% and 0% respectively, of control value. Conduction velocity was reduced significantly by 300, 600 and 2000 microg/ml of EOAZ, at 180 min, to 83.61 +/- 3.28%, 64.06 +/- 8.21% and 22.7 +/- 5.79%, respectively, of control value. All these effects developed slowly and were reversible upon a 180-min wash.     

Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum require automated o-phthalaldehyde derivatization of the drug and immediate injection of the unstable derivatives formed. A new, very sensitive and simple high-performance liquid chromatographic method for quantitation of the drug in human serum using 4-chloro-7-nitrobenzofurazan (NBD-Cl) as a fluorescent labeling agent is presented. In this method the sensitivity was significantly improved and the limit of quantification of 0.002 microg/ml was obtained using 100 microl serum sample and 10 microl injection. However, the LOQ can be improved by increasing the sampling volume. The procedure involved protein precipitation of serum by acetonitrile followed by derivatization with NBD-Cl. Amlodipine was used as internal standard and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The fluorescence derivative of the drug was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.5) containing 1 ml/l triethylamine (65:35, v/v) was used. The calibration curve was linear over the concentration range of 0.002-15 microg/ml. No interferences were found from commonly co-administrated antiepileptic drugs. The method was applied in a randomized cross-over bioequivalence study of two different gabapentin preparations in 24 healthy volunteers.     

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.     

Determination of vancomycin in serum by liquid chromatography-high resolution full scan mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the analysis of vancomycin (VCM) in human serum. The method was based on full scan data with extracted ions for the accurate masses of VCM and the atenolol internal standard obtained by Fourier transform MS. VCM was extracted from serum using strong cation exchange (SCX) solid phase extraction (SPE). The method was found to be linear in the range 0.05-10 microg/ml, which was adequate for quantification of VCM in serum samples, with a limit of quantification (LOQ) of 0.005 microg/ml and a limit of detection (LOD) of 0.001 microg/ml. Intra-day precision (n=5) was +/-3.5%, +/-2.5%, +/-0.7% at 0.05, 0.5 and 5 microg/ml, respectively. Inter-day precision (n=5) was +/-7.6%, +/-6.4%, +/-3.9% at 0.05, 0.5 and 5 microg/ml, respectively. The process efficiency for VCM was in the range 89.2-98.1% with the recovery for the atenolol internal standard (IS) being 97.3%. The method was used to determine VCM levels in patients during peri-operative infusion of the drug, which was found to result in drug levels within the required therapeutic window.     

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.     

Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-beta-naphthylamide was used as the substrate and its hydrolytic product, beta-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 angstroms) reversed-phase C4 column (RPC4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 microl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 microg/ml) and LAP (0.1-46.0 microg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. (n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 microg/ml), and 1.6 pmol/(microl min) (17.5 microg/ml), respectively, with reproducibility of 2-9% R.S.D. (n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. (n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

A HPLC-mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

We report here the validation of an HPLC-electrospray-tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 microl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3'-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm x 2.1mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5-->908.3; internal standard m/z 989.5-->922.3). The assay was linear from 0.5 to 40 microg/l (r2>0.994, n=11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 microg/l) were 93.4-98.2% and <10.7%, respectively (n=10). At the lower limit of quantification (0.5 microg/l) the inter- and intra-day analytical recovery was 94.4-95.8% with imprecision of <14.1% (n=10). The absolute recovery of everolimus (6.5 microg/l) and internal standard (12.5 microg/l) was 96.5 and 88.3%, respectively (n=3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y=0.973x+0.301 (r2=0.986, n=71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.     

High-performance liquid chromatography method with ultraviolet detection for the quantification of the BCR-ABL inhibitor nilotinib (AMN107) in plasma, urine, culture medium and cell preparations

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005-5.0 microg/ml in plasma (r(2)=0.9998) and 0.1-10.0 microg/ml in urine (r(2)=0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27-9.23% in plasma and 1.77-3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 microg/ml in plasma and 0.01 microg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.     

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 degrees C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 microl plasma. Detection limits were 0.1 microg/ml for MPA and AcMPAG, and 2.0 microg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.