Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

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.     

A sensitive and selective LC-MS-MS method for simultaneous determination of picroside-I and kutkoside (active principles of herbal preparation picroliv) using solid phase extraction in rabbit plasma: application to pharmacokinetic study

A rapid, sensitive and selective LC-MS-MS method for the simultaneous quantitation of picroside-I and kutkoside (active constituents of herbal hepatoprotectant picroliv) was developed and validated in rabbit plasma. The analytes and internal standard (Amarogentin) were extracted using Oasis HLB solid phase extraction cartridges. Analysis was performed on Spheri RP-18 column (10 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:MilliQ water (50:50, %v/v) as mobile phase at a flow rate of 1 ml/min with a retention time of 1.39, 1.33 and 1.42 min for picroside-I, kutkoside and amarogentin, respectively. The quantitation was carried out using an API-4000 LC-MS-MS with negative electro spray ionization in multiple reaction monitoring (MRM) mode. The precursor to product ion transitions for picroside-I, kutkoside and amarogentin were m/z 491 > 147, 199; 511 > 167, 235; 585 > 227, respectively. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (within- and between-assay variation), freeze-thaw (f-t), auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.5 ng/ml for both analytes. The recoveries from spiked control samples were > 60 and > 70% for picroside-I and kutkoside, respectively. Accuracy and precision of the validated method were within the acceptable limits of < 20% at low and < 15% at other concentrations. The analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of picroside-I and kutkoside post i.v. bolus administration of picroliv in rabbit.     

A sensitive and selective HPLC/ESI-MS/MS assay for the simultaneous quantification of 16-dehydropregnenolone and its major metabolites in rabbit plasma

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M(1)), 5-pregnene-3beta-ol-20-one (M(2)), 5-pregnene-3beta, 20-diol (M(3)), 5-pregnene-3beta-ol-16, 17-epoxi-20-one (M(4)) and 5,16-pregnadien-3beta, 11-diol-20-one (M(5)) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS-MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M(3) and M(5) where linearity was over the 3.13-400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Development and validation of a liquid chromatography-mass spectrometric method for the determination of DPC 423, an antithrombotic agent,in rat and dog plasma

A sensitive and selective LC-MS-MS method for the determination of DPC 423 (I), an antithrombotic agent, is described. This method used a solid-phase extraction from 0.1 ml plasma with an Isolute C(2) cartridge. HPLC separation was carried out on a YMC ODS-AQ C(18) column (50x2 mm) at a flow-rate of 300 microliter/min with an analysis time of 5 min. Compounds were eluted using a mobile phase of H(2)O/CH(3)CN/HCOOH: 66:34:0.1 (v/v/v), pH 4.0. A structural analogue of I was used as the internal standard to account for variations in recovery and instrument response. Mass spectrometric detection was carried out with a PE Sciex API III(+) triple quadrupole mass spectrometer equipped with a Turbo IonSpray source as the LC-MS interface. Good intra-day and inter-day assay precision (<10% CV) and accuracy (<10% difference) were observed over a concentration range of 0.005-2.5 microM in plasma. The extraction recoveries were approximately 90% and the method was found to be linear for the assay (r(2)>0.999). The method has been successfully applied to discovery and preclinical pharmacokinetic studies, including a dose range-finding study and toxicokinetic exposure studies in rat and dog.     

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

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

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.     

Determination of the substance P receptor antagonist CP-122, 721 in plasma by narrow-bore high-performance liquid chromatography-ionspray tandem mass spectrometry

A simple, highly sensitive and specific LC-MS-MS assay was developed for the determination of CP-122,721 (I) in rat and human plasma. I and a structural analog, CP-129,943 (II, internal standard), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (50:50, v/v). Column effluents were monitored by ionspray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ion combinations of m/z 381 → 205 and 395 → 219 was used to quantitate I and II, respectively. The assay exhibited a linear dynamic range of 0.2–100 ng/ml. Absolute recoveries from plasma were above 80% for both I and II. The precision and accuracy values for the method were within ±3 and ±9%, respectively. Sample analysis times were less than 5 min from one injection to the next. The assay has proved to be applicable to the pharmacokinetic study of I in rats.     

Simultaneous quantification of alpha-/beta-diastereomers of arteether, sulphadoxine and pyrimethamine: a promising anti-relapse antimalarial therapeutic combination, by liquid chromatography tandem mass spectrometry

A rapid, sensitive, selective and specific HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of alpha-/beta-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) in rat blood plasma using propyl ether analogue of beta-arteether as internal standard. The method involved a single-step, liquid-liquid extraction with ethyl acetate and the analytes were chromatographed on a C18 chromatographic column by isocratic elution with methanol:ammonium acetate buffer (10 mM, pH 4) (90:10%, v/v) and analyzed by tandem mass spectrometry. The run time was 4.5 min and the weighted (1/x2) calibration curves were linear over a range of 0.78-400 ng ml-1. The method was validated fully and the lower limit of quantification (LLOQ) in plasma was 0.78 ng ml-1 for all the analytes. The intra- and inter-day precision and accuracy were found to be well within the acceptable limits (<15%) and the analytes were stable after three freeze-thaw (f-t) cycles. The absolute recoveries were consistent and reproducible. The assay method was applied to pre-clinical pharmacokinetic interaction studies of alpha-/beta-AE, SDX and PYR in rats.     

Sensitive method for the determination of ibutilide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid-liquid extraction, and separated on a C(18) column, using acetonitrile-water-10% butylamine-10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20-10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within +/-3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.     

Determination of ragaglitazar,a novel dual acting peroxisome proliferator-activated receptor (PPAR) alpha and -gamma agonist,in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

A sensitive and specific LC/MS/MS method has been developed and validated for determination of ragaglitazar (NNC 61-0029 or DRF 2725) in human plasma. After solid-phase extraction (SPEC((R)) PLUS C(8)) of plasma, separation was performed on a Symmetry Shield RP8 column (mobile phase: acetonitrile: 10 mM ammonium acetate, pH 5.6 (40:60 v/v)). Two ranges were validated having LLOQs of either 0.500 or 100 ng/ml and linearity up to either 500 or 50000 ng/ml. The intra-assay precision and accuracy were 1.1% to 15.7% and 85.8% to 118.2% (range 0.500-500 ng/ml) and 2.0% to 8.8% and 92.9% to 104.8% (range 100-50000 ng/ml). The method was applied for determination of ragaglitazar in plasma from phase 1 and 2 clinical studies.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

A sensitive and selective liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of novel trioxane antimalarials in different biomatrices using sample-pooling approach for high throughput pharmacokinetic studies

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99-357, 99-408 and 99-411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid-liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (< 15%). The average absolute recoveries of 99-357, 99-408 and 99-411 from spiked plasma samples were > 90%, > 70% and > 60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99-357, 99-408 and 99-411 using sample-pooling technique.     

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

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Determination of scutellarin in rat plasma by high-performance liquid chromatography with ultraviolet detection

A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.     

Comparison of a liquid chromatographic method with ultraviolet and ion-trap tandem mass spectrometric detection for the simultaneous determination of sulfadiazine and trimethoprim in plasma from dogs

A method for the simultaneous determination of sulfadiazine and trimethoprim in plasma from Beagle dogs was developed and validated. Samples were deproteinized with acetonitrile and extracted with ethyl acetate. Sulfachloropyridazine and ormethoprim were used as internal standards for the sulfadiazine and trimethoprim analysis, respectively. The chromatography was carried out both on an LC-UV (liquid chromatography-ultraviolet detection) and ion-trap LC-MS(n) (liquid chromatography-mass spectrometric detection) instrument, operating in the positive APCI mode (atmospheric pressure chemical ionization). The purpose of this work was to compare the quantification results of both methods. Both the LC-UV and LC-MS-MS methods were validated for their linearity, accuracy, precision, limit of detection and limit of quantification, according to the requirements defined by the European Community. Calibration curves using plasma fortified between 0.1 and 1 microg/ml of sulfadiazine, 0.1 and 2 microg/ml of trimethoprim, 1 and 20 microg/ml of sulfadiazine showed a good linear correlation (r> or =0.9990, goodness-of-fit< or =8.4%). The results for the accuracy and precision at 1 microg/ml of sulfadiazine and trimethoprim and at 20 microg/ml of sulfadiazine fell within the ranges specified. The limits of quantification of both methods were 0.1 microg/ml. The limits of detection were 0.019 microg/ml of sulfadiazine and 0.024 microg/ml of trimethoprim for the LC-UV method, and 0.020 microg/ml of sulfadiazine and 0.062 microg/ml of trimethoprim for the LC-MS-MS method. The methods have been successfully applied in a pharmacokinetic study to determine the drug concentrations in plasma samples from dogs. A good correlation between the results of both methods was observed (R=0.9724, slope=1.0239, intercept=-0.2080 microg/ml for sulfadiazine and R=0.9357, slope=1.0433, intercept=0.0325 microg/ml for trimethoprim). The precision of both methods was also tested on the results of the same samples using an F-test (alpha=0.05), indicating that both methods did not differ in precision.     

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.