Method for the quantitative analysis of cilostazol and its metabolites in human plasma using LC/MS/MS

An LC/MS/MS method for the simultaneous determination of cilostazol, a quinolinone derivative, and three active metabolites, OPC-13015, OPC-13213, and OPC-13217, in human plasma was developed and validated. Cilostazol, its metabolites, and the internal standard, OPC-3930 were extracted from human plasma by liquid-liquid partitioning followed by solid-phase extraction (SPE) on a Sep-Pak silica column. The eluent from the SPE column was then evaporated and the residue reconstituted in a mixture of methanol/ammonium acetate buffer (pH 6.5) (2:8 v/v). The analytes in the reconstituted solution were resolved using reversed-phase chromatography on a Supelcosil LC-18-DB HPLC column by an 17.5-min gradient elution. Cilostazol, its metabolites, and the internal standard were detected by tandem mass spectrometry with a Turbo Ionspray interface in the positive ion mode. The method was validated over a linear range of 5.0-1200.0 ng/ml for all the analytes. This method was demonstrated to be specific for the analytes of interest with no interference from endogenous substances in human plasma or from several potential concomitant medications. For cilostazol and its metabolites, the accuracy (relative recovery) of this method was between 92.1 and 106.4%, and the precision (%CV) was between 4.6 and 6.5%. During the validation, standard curve correlation coefficients equalled or exceeded 0.999 for cilostazol and its metabolites. These data demonstrate the reliability and precision of the method. The method was successfully cross-validated with an established HPLC method.     

The quantitative determination of cilostazol and its four metabolites in human liver microsomal incubation mixtures by high-performance liquid chromatography

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150 x 4.6 mm, 5 microm) column and a Cosmil C-18 column (150 x 4.6 mm, 5 microm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile in acetate buffer (pH = 6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43-17.59. The components of liver microsomal incubation mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml(-1) for cilostazol and 100 ng ml(-1) for each of the metabolites. This method has been validated for linear ranges of 100-4000 ng ml(-1) for OPC-13213, OPC-13217 and OPC-13326; 100-2000 ng ml(-1) for OPC-13015; and 1000-20000 ng ml(-1) for cilostazol. The percent relative recovery of this method was established to be 81.2-101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8-7.7%. The autosampler stability of the analytes was evaluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, accurate and reproducible.     

Direct-injection HPLC assay for the determination of a new carbapenem antibiotic in human plasma and urine

Reversed-phase high-performance liquid chromatography (RP-HPLC) assays using ultraviolet (UV) absorbance detection have been developed for the determination of a new carbapenem antibiotic I in human plasma and urine. A column-switching technique is employed in the HPLC methods to perform on-line extraction and separation for each sample. Each plasma sample is thawed, centrifuged, stabilized, and then injected onto an in-line reversed-phase extraction column using a methanol (8%)/phosphate buffer, pH 6.5. After 3 min, the analytes are back-flushed off the extraction column with a mixture of acetonitrile (5.5%) and methanol (10%)/phosphate buffer (pH 6.5) for 3 min onto a BDS Hypersil 3 microm C18 (100 x 4.6 mm i.d.) analytical column. The sample preparation and HPLC conditions for the urine assay are similar to the plasma assay, except that a CN extraction column is used. Both assays are specific with respect to endogenous material and the major metabolite II, and both are linear over the concentration range of 0.25-50, and 2-200 microg/ml, respectively. The assays were successfully applied to a clinical dose-ranging study. One limitation of the on-line extraction method is that the extraction column needs to be replaced regularly every 100-150 plasma samples and every 200-300 urine samples. Subsequently, the urine method was modified to an ion-pair HPLC assay for the simultaneous determination of both the antibiotic I and its metabolite II.     

Hydrolytic Degradation Profile and RP-HPLC Estimation of Cilostazol in Tablet Dosage Form

A simple, selective, precise and stability-indicating high-performance liquid-chromatographic method of analysis of cilostazol in pharmaceutical dosage form was developed and validated. The solvent system consisted of 10 mM phosphate buffer (pH 6.0):acetonitrile:methanol (20:40:40). Retention time of cilostazol in C18 column was 5.7 ± 0.1 min at the flow rate 1.3 ml/min. Cilostazol was detected at 248 nm at room temperature. The linear regression analysis data for the calibration plots showed good linear relationship with correlation coefficient value, r ² =0.9998 in the concentration range 100–3200 ng/ml with slope 43.45 intercept 156.75. The method was validated for linearity, range, accuracy, precision and specificity. Cilostazol was determined in tablet dosage form in range of 99.58-100.67% with 0.4600 standard deviation. Stress studies were conducted in acid and alkali hydrolysis with gradual increasing concentration. Cilostazol was found to be stable in various concentrations of acidic and alkaline.     

Validated high performance liquid chromatographic method for simultaneous determination of rosiglitazone, cilostazol, and 3,4-dehydro-cilostazol in rat plasma and its application to pharmacokinetics

A high performance liquid chromatographic (HPLC) method for simultaneous determination of rosiglitazone, CAS 122320-73-4, RSG), cilostazol (CAS 73963-72-1, CLZ) and its active metabolite 3, 4-dehydro-cilostazol (DCLZ), using pioglitazone (PIO) as internal standard (IS), in rat plasma is described. The plasma was extracted with methyl t-butyl ether, the dry extract was reconstituted in mobile phase and the aliquot was injected. The eluent drugs were detected by UV at dual wavelength of 226 nm (RSG and DCLZ) and 257 nm (CLZ). The mobile phase consisting of acetonitrile:potassium di-hydrogen phosphate buffer (35:65 v/v) was used at the flow rate of 1.2 ml/min on a reverse phase C18 column. The absolute recovery was above 90% of all analytes over the concentration range of 25-2500 ng/ml for RSG and CLZ and 20-2000 ng/ ml for DCLZ. The relative standard deviation (RSD) of the inter-day and intra-day precision ranged from 2.8 to 8.4% and 0.9 to 5.9%, respectively. The method is simple, rapid, accurate and sensitive and was applied to pharmacokinetic studies.     

Liquid chromatography - tandem mass spectrometry for the simultaneous quantitation of glipizide, cilostazol and its active metabolite 3, 4-dehydro-cilostazol in rat plasma: application for a pharmacokinetic study

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous quantitation of glipizide, cilostazol and 3, 4-dehydro-cilostazol in rat plasma was developed and validated. Glimepride was used as an internal standard (IS). The analytes were extracted by using liquid-liquid extraction procedure and separated on a reverse phase C18 column (50 mm×4.6 mm i. d., 5 μ) using acetonitrile: 2 mM ammonium acetate buffer, pH 3.2 (90:10, v/v) as mobile phase at a flow rate 0.4 mL/min in an isocratic mode. Selective reaction monitoring was performed using the transitions m/z 446.4>321.1, 370.2>288.3, 368.3>286.2, and 491.4>352.2 to quantify glipizide, cilostazol, 3, 4-dehydro-cilostazol and glimepride, respectively. Calibration curves were constructed over the range of 25-2 000 ng/mL for glipizide, cilostazol and 3, 4-dehydro-cilostazol. The lower limit of quantitation was 25 ng/mL for all the analytes. The recoveries from spiked control samples were>76% for all analytes and internal standard. Intra and inter day accuracy and precision of validated method were within the acceptable limits of at all concentration. The quantitation method was successfully applied for simultaneous estimation of glipizide, cilostazol and 3, 4-dehydro-cilostazol in a pharmacokinetic drug-drug interaction study in wistar rats.     

反相HPLC法同时测定人尿中茶碱及其两种代谢产物

目的:建立一种同时测定人尿中茶碱及其1,3-二甲基尿酸(1,3-DMU)和3-甲基噻嗪(3-MX)代谢产物的HPLC方法.方法:尿样用异丙醇/二氯甲烷(2/8)混合液提取,有机相在空气吹干,用流动相复溶后进行HPLC分析.色谱柱为Diamonsil ODS C_(18)5 μm,150 mm×4.6 mm I.D),流动相由0.1%甲酸液和乙腈(95:5)组成,流速1.0 mL/min,测定波长280 nm.测定12名受试者单剂量和多剂量口服茶碱后24 h内尿中茶碱及其代谢物累计排泄量.结果:尿中茶碱及其代谢物1,3 DMU和3-MX的线性范围分别为0.312～40.0、0.156～20.0、0.078～10.μg/mL,最低可定量浓度分别为0.312、0.156、0.078μg/mL.批间和批内的变异小于15%,回收率大于70%.结论:该方法的特异性、灵敏度能够满足临床上对人尿中茶碱及其代谢产物同时测定的要求.     

Development and validation of a high-performance liquid chromatography assay and a capillary electrophoresis assay for the analysis of adenosine and the degradation product adenine in infusions

A high-performance liquid chromatography (HPLC) method and a capillary electrophoresis (CE) method for the analysis of adenosine and the degradation product adenine in infusion solutions have been developed and validated. The HPLC separation of the analytes was achieved on a RP-18 column, using a mobile phase, consisting of 20mM ammonium acetate, pH 6.0, containing 5% of acetonitrile at a flow rate of 1ml/min. Thymidine was used as internal standard. The CE separation was performed in a fused-silica capillary with a 100mM sodium phosphate buffer, pH 2.7, at an applied voltage of 25kV, using cytidine as internal standard. The assays were validated with regard to linearity, range, limit of detection (LOD), limit of quantitation (LOQ), specificity, and precision. Both methods were specific allowing reliable quantification of the analytes. Compared to the CE method, HPLC analysis yielded a two- to five-fold lower LOD. With respect to analysis time, CE was faster than HPLC. The applicability of both methods for the determination of the purity and stability of adenosine in the infusion solutions is demonstrated.     

Simultaneous determination of mycophenolic acid and its glucuronides in human plasma using isocratic ion pair high-performance liquid chromatography

Therapeutic drug monitoring (TDM) of mycophenolic acid (MPA) following administration of mycophenolate mofetil (MMF) or the enteric-coated sodium salt of MPA formulations, seems beneficial because of the large intra- and inter-individual variability in MPA pharmacokinetics. MPA is an active component from these oral formulations and are further metabolised to inactive phenolic glucuronide (MPAG) and active acyl glucuronide (AcMPAG). This study aims to determine simultaneously these three metabolites of MMF using isocratic ion pair HPLC and to evaluate the short-term stability of AcMPAG in human plasma. Samples were prepared using solid phase extraction. Chromatographic separation was achieved over an RP column (TSKgel ODS-80Ts, 150 mm x 4.6 mm i.d., 5 microm particle size) with acetonitrile and 30 mM tetra-n-butylammonium bromide containing 5 mM ammonium acetate at pH 9.0 (33/67, v/v) as the mobile phase. The flow rate of the mobile phase was 1ml/min, and the wavelength of determination by UV detection was 250 nm (run time, 16 min). Calibration curves for MPA, MPAG and AcMPAG in human plasma were linear over a concentration range of 0.05-50, 0.1-400 and 0.08-8 microg/ml, respectively. Intra- and inter-assay R.S.D. were<6.5%. Extraction efficiencies were more than 85% for all analytes. Since AcMPAG was unstable in human plasma, plasma acidification was needed for the quantification of AcMPAG. Large interindividual variability was observed in the AcMPAG pharmacokinetics in the early period after renal transplantation. In conclusion, a simple, accurate and reproducible HPLC method to measure simultaneously these three MMF metabolites has been established. The method will be helpful in evaluating pharmacokinetics of MPA and its glucuronides.     

Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.     

Determination of berberine in human plasma by liquid chromatography-electrospray ionization-mass spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the determination of berberine in human plasma using chlorobenzylidine as the internal standard (IS) has been developed and validated. The plasma samples were prepared by LLE and the analytes were chromatographically separated on a Hanbon Lichrospher 5-C18 HPLC column under gradient elution with a mobile phase consisted of acetonitrile and 10mm ammonium acetate buffer containing 0.1% formic acid. Berberine was determined with electrospray ionisation-mass spectrometry (ESI-MS). LC-ESI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at M(+)m/z 336.1 for berberine and M(+)m/z 464.1 for the IS. Calibration curve was linear over the range of 0.020-3.0 ng/ml. The lower limit of quantification (LLOQ) was 0.020 ng/ml. The intra- and inter-run variability values were less than 6.7 and 7.7%, respectively. The method has been successfully applied to determine the plasma concentration of berberine in healthy Chinese volunteers.     

高效液相色谱法测定人血浆中西洛他唑的浓度

目的 :建立以高效液相色谱法测定人血浆中西洛他唑浓度的定量分析方法。方法 :色谱柱为迪马公司C18(150mm×4 6mm ,5μm ) ,流动相为乙腈 -水 (40∶60 ,V/V ) ,流速为1 4ml/min ,检测波长为254nm。结果 :西洛他唑检测浓度在25～2000ng/ml范围内线性关系良好 (r=0 9999),最低检测浓度为12 5ng/ml ,回收率为99 .99 %～101 .44 % ,日内、日间相对标准差分别为0 .20 %～2 90 %、0. 19 %～2. 13 % (n=5)。结论 :本方法简便、快捷、灵敏、准确 ,可用于西洛他唑人体药动学研究。     

HPLC determination of D and L moxalactam in human serum and urine

A high-pressure liquid chromatographic procedure was developed to determine the D and L isomers of moxalactam in human plasma and urine. After protein precipitation with hydrochloric acid the sample was extracted with ethyl acetate. It was then back extracted into tromethamine buffer (pH 8.0) and washed with octanol. Extraction recovery from plasma ranged from 73-81%. An aliquot of the tromethamine buffer was then injected onto a C18-muBondapak column. The mobile phase was 3% acetonitrile in 0.05 M ammonium acetate pH 6.5 buffer. Samples were quantitated by UV detection at 275 nm and 0.01 aufs. The lower limit of detection was 0.5 microgram/ml for each isomer. Preliminary stability studies were performed to assess proper sample handling and storage conditions. The procedure was evaluated in a clinical setting to demonstrate its applicability to the study of moxalactam pharmacokinetics in critically ill patients.     

Resolution of salbutamol enantiomers in human urine by reversed-phase high performance liquid chromatography after derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glycopyranosyl isothiocyanate

A stereospecific HPLC method has been developed for the resolution of the enantiomers of salbutamol in human urine. After solid-phase extraction and derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate, the diastereomeric derivatives were resolved (Rs=1. 83) on 5 μm octadecylsilan column using 35% acetonitrile in 0.05M ammonium acetate buffer (pH=6) as a mobile phase with electrochemical detection. The diastereomeric derivatives were formed within 30 min. The detection limit of each enantiomer was 20 ng/ml (S/N=3).     

Determination of linezolid in human serum and urine by high-performance liquid chromatography.

An HPLC method is described for the determination of the new oxazolidinone antibiotic linezolid (I) in human biofluids. After precipitation of serum proteins with perchloric acid the protein free supernatant was separated by isocratic reversed-phase chromatography on a Nucleosil-100 5C18 column. The mobile phase consisted of a mixture of acetonitrile: sodium acetate buffer: water (180:100:720, v/v) adjusted to pH 3.7. Urine was diluted with aqueous buffer solution. The column eluate was monitored at 250 nm. Validation of the method yielded satisfactory results for serum (and urine); detection limit 0.07 mg/l (2.4), lower limit of quantitation 0.14 mg/l (4.7), linear range 20 mg/l (500), imprecision within series (c.v.) 1.8-2.5% (0.8-1.0), imprecision between series (c.v.) 1.8-9.3 (0.4-9.3), recovery 99-102% (93-103). Comparison of HPLC results with results obtained using a quantitative microbiological assay yielded acceptable agreement both for serum and urine. The method was successfully used in a pharmacokinetic study with human volunteers.     

Buspirone, fexofenadine, and omeprazole: quantification of probe drugs and their metabolites in human plasma

Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a "cocktail". This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5'-hydroxyomeprazole; buspirone and its metabolite 1-[2-pyrimidyl]-piperazine (1PP); and fexofenadine. These analytes and the internal standard lansoprazole were extracted from plasma using protein precipitation with acetonitrile. Gradient reverse-phase chromatography was performed with 7.5mM ammonium bicarbonate and acetonitrile, and the analytes were quantified in positive ion electrospray mode with multiple reaction monitoring. The method was validated to quantify the concentration ranges of 1.0-1000ng/ml for omeprazole, omeprazole sulfone, 5'-hydroxyomeprazole, and fexofenadine; 0.1-100ng/ml for buspirone, and 1.0-100ng/ml for 1PP. These linear ranges span the plasma concentrations for all of the analytes from probe drug studies. The intra-day precision was between 2.1 and 16.1%, and the accuracy ranged from 86 to 115% for all analytes. Inter-day precision and accuracy ranged from 0.3 to 14% and from 90 to 110%, respectively. The lower limits of quantification were 0.1ng/ml for buspirone and 1ng/ml for all other analytes. This method provides a fast, sensitive, and selective analytical tool for quantification of the six analytes in plasma necessary to support the use of this probe drug cocktail in clinical studies.     

Determination of depleted uranium, pyridostigmine bromide and its metabolite in plasma and urine following combined administration in rats

A simple and reliable method was developed for the quantification of depleted uranium, the anti nerve agent drug pyridostigmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The method involved using solid phase extraction and spectrophotometric determination of uranium, and high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 280 nm for PB and its metabolite. Uranium was derivatized using dibenzoylmethane (DBM) then the absorbance was measured at 405 nm. PB and its metabolite were separated using a gradient of 1--40% acetonitrile in 0.1% triflouroacetic acid water solution (pH 3.2) at a flow rate of 0.8 ml/min in a period of 14 min. Limits of detection were 2 ng/ml for uranium and 50 ng/ml for PB and its metabolite. Limits of quantitation were between 10 and 100 ng/ml for uranium and the other two analytes, respectively. Average percentage recovery of five spiked plasma samples were 83.7+/-8.6, 76.8+/-6.7, 79.1+/-7.1, and from urine 82.7+/-8.6, 79.3+/-9.5 and 78.0+/-6.2, for depleted uranium, PB and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear for standards between 100 and 1000 ng/ml for all three analytes. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.     

Quantitative determination of a novel insulin sensitizer and its para-hydroxylated metabolite in human plasma by LC-MS/MS

I, 5-[3-[3-(4-phenoxy-2-propylphenoxy)-propoxy]-phenyl]-2,4-thiazolidinedione sodium salt, is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist for potential use in diabetic patients. The compound has a para-hydroxylated metabolite, II, which has also been shown to exhibit PPAR activity. An LC-MS/MS method for the simultaneous determination of I and its active metabolite (II) in human plasma has been successfully developed. The method consists of treating 0.5 ml plasma with ammonium acetate (pH 9.6; 50mM) and extracting I, II and internal standard (III, Fig. 2) with 5 ml ethyl acetate. The ethyl acetate is evaporated and the samples are reconstituted in 0.1 ml acetonitrile:0.1% formic acid (65:35, v/v). The entire extraction procedure, as well as sample collection, was performed in glass tubes and vials to overcome the analytes adherence to polypropylene. A linear HPLC gradient was used to separate the analyte, metabolite, internal standard, and other interfering, non-quantitated metabolites. Detection was by negative ionization MS/MS on a turbo ionspray probe. Precursor-->product ion combinations were monitored in multiple reaction monitoring (MRM) mode. The linear range is 0.05-20 ng/ml for I and 0.1-20 ng/ml for II. Recoveries were 59.4, 90.1 and 56.8% for I, II and III, respectively. Intraday variation using this method was <==7.0% for I and <==9.2% for II. The method exhibits good linearity and reproducibility for each analyte and good sensitivity, selectivity and robustness when used for the analysis of clinical samples.     

HPLC-MS/MS法测定人血浆中西洛他唑浓度

目的建立灵敏快速检测人血浆中西洛他唑浓度的HPLC-MS/MS方法。方法采用色谱柱为Thermo Hy-purity C18（150 mm×2.1 mm,5μm,USA）,流动相为乙腈（含0.1%甲酸）-甲醇=25∶75（v/v）;流速为0.2mL.min-1;ESI＋离子源,MRM进行离子方式监测。质谱参数：源电压3 500V,源温度100℃,去溶剂化温度350℃,锥孔气流速50 L.h^-1,去溶剂化气流速350 L.h^-1;血浆处理采用乙腈直接沉淀萃取方法。结果血浆中西洛他唑检测方法的线性范围为4.90-2 510 ng.mL-1,最低检出浓度为4.90 ng.mL-1,血浆中西洛他唑的方法回收率在80%-120%,日内RSD〈10%,日间RSD〈15%。结论本方法用于测定人血浆中西洛他唑的浓度,简单、快速、灵敏。