A one-step enzyme-linked immunosorbent assay for a novel osteoblast differentiation-promoting compound, TAK-778 in serum

TAK-778, ((2R,4S)-(-)-N-[(4-diethoxyphosphorylmethyl)phenyl]- 1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2- carboxamide), is a novel osteoblast differentiation-promoting compound, and its sustained-release formulation is expected to be clinically used for the enhancement of fracture healing. To date, TAK-778 levels in serum have been measured using conventional reverse-phase HPLC with inferior sensitivity and time-consuming procedures. We have produced polyclonal antibodies against TAK-778 by using one of its derivatives coupled with a carrier protein, and developed a one-step ELISA for the determination of TAK-778 in serum. The antibodies had minimal cross-reactivities to the biologically inactive metabolites including the oxidized-form (0.36%) and the cleavaged-form (0.00083%). The competitive ELISA was accomplished within three hours with a detection limit of 0.5 ng/ml serum, and the coefficients of variations for samples ranging from 1 ng/ml to 80 ng/ml were 1.1-4.4% in an intra-assay and 3.1-9.6% in an inter-assay. The present ELISA is so rapid, sensitive and selective for TAK-778 that it could be conveniently used in a clinical field for the determination of many serum specimens.     

Sustained-release microcapsules of a bone formation stimulant, TAK-778, for local injection into a fracture site.

The benzothiepin derivative (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) is a potent bone formation stimulant. A sustained release formulation was prepared by encapsulating the drug into biodegradable microcapsules for local application to fracture repair in rats. The microcapsules consisted of TAK-778 (10% w/w) and a biodegradable polymer, copoly (d,l-lactic/glycolic acid), with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 15 k. The TAK-778 amount at the injection site progressively diminished for 4 weeks after administration, and the serum level of TAK-778 was sustained over the same period. The local concentration of TAK-778 after administration of the microcapsules was simulated by a two-compartment open model. In the model, a first-order release rate constant and a transfer rate constant were obtained from the release profile of the microcapsules and the serum level of TAK-778 after administration of the TAK-778 solution, respectively. Localization at the injection site was examined by radiography using microcapsules in which iodoform was encapsulated as a contrast agent. The microcapsules formed a clot at the injection site, and their spread was narrowly restricted. The local concentration was calculated to be maintained within the range 10(-3)-10(-6) M over 4 weeks on the assumption that the dose and spread volume were 5 mg of TAK-778/site and 3 mL, respectively.     

Development and validation of a sensitive LC-MS/MS method for the determination of adefovir in human serum and urine

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of adefovir (PMEA) in human serum and urine. The analyte was separated on a Diamonsil C(18) column (250 mm x 4.6 mm i.d., 5 microm particle size) by isocratic elution with methanol-water-formic acid (20:80:0.1, v/v/v) at a flow rate of 0.6 ml/min, and analyzed by mass spectrometry in multiple reaction-monitoring mode. The precursor-to-product ion transitions of m/z 274-->162 and m/z 226-->135 were used to measure and quantify the analyte and internal standard (I.S.), respectively. The weighted (1/x(2)) calibration curve was linear over serum concentration range 1.25-160.00 ng/ml and urine concentration range 0.05-8.00 microg/ml, with a correlation coefficient (r) of 0.9992 and 0.9978, respectively. The lower limit of quantification in human serum was 1.25 ng/ml. The inter- and intra-day precisions (R.S.D.%) in both serum and urine were lower than 8.64%, the mean method accuracies and recoveries from spiked serum samples at three concentrations ranged from 96.3 to 102.0% and 56.5 to 59.3%, respectively. The serum extract was stable when stored for 24h. The developed method was successfully applied to determine PMEA in human serum and urine, and proved suitable to clinical pharmacokinetic study.     

Simultaneous determination of clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients

A liquid chromatography tandem mass spectrometry (LC-MS-MS) assay method for the simultaneous determination of clozapine and its N-desmethyl (norclozapine) and N-oxide metabolites in human plasma is described. The compounds were extracted from plasma by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using positive ion atmospheric pressure electrospray ionization method by a TurboIonspray source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 327 --> m/z 270 for clozapine, m/z 313 --> m/z 192 for norclozapine, m/z 343 --> m/z 256 for clozapine-N-oxide and m/z 421--> m/z 201 for internal standard. The standard curves of clozapine, norclozapine and clozapine-N-oxide were linear over the range of 1 ng/ml to 1000 ng/ml when 0.5 ml of plasma was used for the analysis (r(2) >0.998). Three pooled plasma samples collected from patients who were treated with clozapine were used as long-term quality control samples to check the validity of spiked standard curve samples made at various times. The intra- and inter-assay variations for the spiked standard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The LC-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific, sensitive, accurate and rapid. This method is currently being used for the plasma level monitoring of clozapine and its N-desmethyl and N-oxide metabolites in patients treated with clozapine. The plasma levels of clozapine, norclozapine and clozapine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 58%+/-14% and 17%+/-6% of clozapine concentrations in plasma, respectively.     

Simultaneous determination of five HIV protease inhibitors nelfinavir,indinavir, ritonavir,saquinavir and amprenavir in human plasma by LC/MS/MS

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been developed to measure the levels of five HIV protease inhibitors nelfinavir (NFV), indinavir (IDV), ritonavir (RTV), saquinavir (SQV) and amprenavir (APV) in human plasma. The analytes and internal standard are isolated from plasma by a simple acetonitrile precipitation of plasma proteins followed by centrifugation. LC-MS-MS in positive mode used pairs of ions at m/z of 568.4/330.0, 614.3/421.2, 720.9/296.0, 671.1/570.2 and 505.9/245.0 for NFV, IDV, RTV, SQV and APV, respectively and 628/421 for the internal standard. Two 1/x weighted linear calibration curves for each analyte were established for quantitation with the low curve ranging from 5 to 1000 ng/ml and while the high curve ranging from 1000 to 10,000 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the ranges of the standard curves were less than 10%. The overall recovery of NFV, IDV, RTV, SQV and APV were 88.4, 91.4, 92.2, 88.9 and 87.6%, respectively.     

LC-MS-MS determination of brostallicin in human plasma following automated on-line SPE

LC-MS-MS method using automated on-line solid-phase extraction (SPE) has been developed and validated for the quantitation of brostallicin (I), a new distamycin derivative, in human plasma. I is a DNA minor groove binder currently under phase I-II clinical evaluation as an anticancer drug. Plasma (0.4 ml) was spiked with 0.2 ml stable label IS solution and placed in a 96-well plate maintained at +4 degrees C. Aliquots of 0.1 ml of prepared samples were loaded into the on-line SPE HySphere Resin SH cartridges (10 mm x 2 mm ID) and the analytes back eluted with the mobile phase into LC-MS-MS system. A Platinum Cyano column (100 mm x 4.6 mm, 3.6 microm) was used to perform the chromatographic analysis. The mobile phase was acetonitrile-ammonium formate buffer (pH 3.5; 20 mM) (70:30, v/v) at a flow rate of 1.0 ml/min. LC flow was split so that 300 microl/min was directed toward the mass spectrometer interface. Retention time of I was 2.6 min and the total cycle time was 8 min. MS detection used an Applied Biosystems/MDS SCIEX API 365 with a TurboIonSpray interface and MRM (m/Z: 725/257 for I and m/Z: 729/257 for IS) operated in positive ion mode. The method was validated over the calibration range 0.124-497 ng/ml. A negligible carry-over effect from the system was observed. In spite of the known instability of I in human plasma (about 20% decrease over 12 h), the ratio analyte/IS peak area showed good stability over the analysis time required for 96 samples. The automated on-line SPE method can be considered as a valid alternative to the off-line manual SPE procedure previously developed.     

An LC-MS-MS method for the determination of indinavir, an HIV-1 protease inhibitor, in human plasma

A method for the determination of indinavir (IDV) (L-735 524) in human plasma by LC-MS-MS is discussed, and the validation data is presented. The analyte and internal standard are isolated from plasma by a simple acetonitrile precipitation of plasma proteins followed by centrifugation. LC-tandem mass spectrometry in positive ion, multiple reaction monitoring mode used pairs of ions at m/z of 614/421 for indinavir and 628/421 for internal standard, respectively. The calibration curve had a linear range from 3.0 to 12320 ng/ml when linear least square regression weighing 1/x was applied to the concentration versus peak area plot. The advantages of this method are the fast sample preparation, wide dynamic assay range and quick analysis taking only 5 min for each sample run. The robust nature of this assay has been further verified during routine use over several months involving multiple analysts.     

Trace-level determination of nifedipine in human serum by reversed phase high performance liquid chromatography

A rapid, simple, specific and sensitive high performance liquid chromatographic method has been developed for the determination of nifedipine (adalate) in human serum using diazepam (valium) as an internal standard (I.S.). The method utilized a 5 microm nonpolar C18 reversed phase Hypersil (ODS) column (250 x 3 mm i.d.). The mobile phase consisted of 60% v/v, acetonitrile in water, adjusted to pH 3.7 with glacial acetic acid and ammonium acetate, and pumped at a flow rate of 2 ml/min. The effluent was monitored by UV detection at 340 nm and a sensitivity fixed at 0.02 aufs. Each analysis required no longer than 4 min. The minimum detectable amount of nifedipine in serum was 3 ng/ml, and the mean absolute recovery was 93.5%. The within and between-day coefficients of variation at three different concentrations from 15-160 ng/ml ranged from 2.07 to 7.76%, and from 3.15 to 7.98% respectively. Calibration graphs for 10 to 200 ng/ml were linear with a mean correlation coefficient, r (n = 36) of 0.9991. The method was validated for accuracy, sensitivity, selectivity and reproducibility and finally was utilized and proved to be suitable in a bioavailability study of two products of nifedipine following oral administration to healthy male subjects.     

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

A sensitive and specific method for the quantification of sufentanil in human plasma by liquid chromatography coupled with tandem mass spectrometry has been developed. Fentanyl was used as the internal standard. Rapid sample preparation involved purification on a C(18) solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C(18) mu-HPLC column. LC-MS-MS detection was performed by atmospheric pressure ionisation (API) source equipped with an ionspray (IS) interface operating in the positive ion mode. For unambiguous substance confirmation, three analyte precursor-product ion combinations were monitored during multiple reaction monitoring (MRM) LC-MS-MS analysis. The method's performance characteristics were evaluated in blank and spiked control plasma samples. Overall accuracy (relative error, R.E., %), repeatability (relative standard deviations, R.S.D., %) and within-laboratory reproducibility (R.S.D., %) ranged from -9.28 to -2.71%, from 6.42 to 2.82% and from 13.52 to 6.06%, respectively, for sufentanil. The limit of quantification for sufentanil in human plasma samples was 0.3 ng ml(-1). Due to its high sensitivity and specificity, the method was successfully employed for sufentanil determination in maternal plasma samples collected immediately before epidural administration of a single sufentanil dose to women in labour, 20 min after drug administration, and at birth in arterial and venous umbilical cord plasma samples from the newborn babies. Research is in progress to adopt the method for performance of complete pharmacokinetic studies of sufentanil in human plasma.     

The determination of minoxidil in human serum by high-performance liquid chromatography with amperometric detection

An ion-pairing reversed-phase HPLC assay employing amperometric detection has been developed for the determination of free minoxidil (MNX) in human serum. The drug is isolated from the serum and concentrated by solid phase extraction with a disposable cartridge column containing ethylsilane (C2) bonded phase packing material. The average absolute recovery from serum was 85%. The HPLC separation is performed on a Sperisorb-C(8) column, using n-octanesulphonic acid as the pairing ion. The method exhibits linear behaviour from 0.3 to 100 ng/ml for spiked serum samples. The average daily relative standard deviations of replicate samples at the 0.75 and 2.0 ng/ml levels were 9.6 and 6.4%, respectively. Utilizing a 1 ml sample the limit of detection (S/N 3) was 0.3 ng/ml.     

LC-MS-MS determination of nemorubicin (methoxymorpholinyldoxorubicin,PNU-152243A) and its 13-OH metabolite (PNU-155051A) in human plasma

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of nemorubicin, (PNU-152243A, 3'-deamino-3'[2(S)-methoxy-4-morpholinyl]doxorubicin) hydrocloride and its reduced metabolite PNU-155051 in human plasma has been developed and validated. The method involved solid phase extraction (SPE) in 96-well plates. Plasma samples (0.5 ml plasma, spiked with doxorubicin as internal standard and diluted with 0.5 ml of 0.01 M borate buffer, pH 8.4) were extracted using Oasis HLB SPE material. The elution of PNU-152243, PNU-155051 and of IS was performed with 1 ml of methanol:0.1 M formic acid mixture (90:10, v/v). The organic phase was reduced to dryness under a stream of nitrogen at 20 degrees C and the residue was reconstituted with 0.25 ml of 10 mM ammonium formate buffer pH 4.15:acetonitrile mixture (90:10, v/v). Aliquots of 60 microl of the resulting solution were injected onto the LC-MS-MS system. A Zorbax SB C18 column (2.1 x 150 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 10 mM pH 4.15:acetonitrile (73:27, v/v) with a flow-rate of 0.2 ml/min. Detection was achieved by a PE-SCIEX API 3000 with Turbo IonSpray interface, and multiple reaction monitoring (645 --> 321 for PNU-152243, 647 --> 363 for PNU-155051 and 545 --> 345 m/z for doxorubicin) operated in positive ion mode. A weighted linear regression was used to calculate PNU-152243 and PNU-155051 concentrations in QC and unknown samples. Linearity, precision, accuracy and recovery of the method were evaluated over the concentration range of 0.1-5 ng/ml for both compounds. No interference from blank human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from patients who had received a single intrahepatic artery dose of PNU-152243A.     

Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry

A highly selective and sensitive HPLC-ESI-MS-MS method was developed for the determination of oleanolic acid in human plasma. The oleanolic acid and glycyrrhetinic acid (internal standard) were recovered from plasma with ethyl acetate liquid-liquid extraction. The organic extracts were dried under a stream of warm nitrogen, reconstituted in mobile phase and injected into a Zorbax-Extend ODS analytical column (150 mm x 4.6 mm i.d., 5 microm), with the mobile phase consisting of methanol-ammonium acetate (32.5 mM) (85:15, v/v) pumped at a flow rate of 1.0 ml/min, and 30% of the eluent was split into a MS system with electrospray ionization tandem mass (ESI-MS-MS) detection in negative ion mode. The tandem mass detection was performed on a Finnigan Surveyor LC-TSQ Quantum Ultra AM tandem mass spectrometer operated in selected reaction monitoring mode. The parent to product ion combinations of m/z 455.4-->455.4 and 469.3-->425.2 at 38 V 1.5 mTorr Ar CID were used to quantify oleanolic acid and glycyrrhetinic acid, respectively. The assay was validated in the concentration range of 0.02-30.0 ng/ml for oleacolic acid when 0.5 ml of plasma was processed. The precision of the assay (expressed as relative standard deviation, R.S.D.%) was less than 15% at all concentrations levels within the tested range and adequate accuracy, and the limit of quantification was 0.02 ng/ml. The established method was applied for the pharmacokinetics study of oleanolic acid capsules in 18 healthy male Chinese volunteers with the mean values of C(max), T(max), AUC(0-48), AUC(0-infinity), t(1/2,) CL/F, and V/F of oleanolic acid after p.o. a single 40 mg dose obtained were 12.12 +/- 6.84 ng/ml, 5.2 +/- 2.9h, 114.34 +/- 74.87 ng h/ml, 124.29 +/- 106.77 ng h/ml, 8.73 +/- 6.11 h, 555.3 +/- 347.7 L/h, and 3371.1 +/- 1,990.1 L, respectively.     

HPLC-MAXPLOT UV检测法测定血清中丹那唑及其代谢产物

本文建立了同时测定人体血清中丹那唑(Ⅰ)及其主要代谢产物2-羟甲基妊娠素(Ⅱ)和其它代谢产物妊娠素(Ⅲ)的高效液相色谱法。方法用YWG C18柱并以甲醇—水(74:26)作为流动相,应用紫外最大吸收作图法(maxplot)检测技术,以UV 285,240nm分别测定Ⅰ,Ⅱ和Ⅲ。使原型药和代谢产物都达到较高检测灵敏度。同时对方法的专一性、准确性和精密度等方面进行了评价,并应用于临床监测。     

高效液相色谱法测定血清及尿中的速尿含量

本文通过简单的预处理,应用高效液相色谱法进行了血清及尿中的速尿含量测定。本法简便、快速、重现性好。采用了血清以乙腈除蛋白、尿稀释50倍后测定的预处理法。高效液相色谱条件为:反相柱,以含30%乙腈的5mM 己烷磺酸钠水溶液作流动相(pH3.5),流速2.5ml/min。使用荧光检测器,激发波长333nm、荧光检测波长415nm。血清及尿中速尿的回收率分别为93.5—101.7%及90.8%。重现性也得到良好的结果,用血清测定时,日内变异系数为3.48%,用尿测定时,日内变异系数为2.58%。     

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.     

Selective,sensitive, and rapid liquid chromatography-tandem mass spectrometry method for determination of Guaifenesin in human plasma

A simple, sensitive, selective, and rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for quantitation of guaifenesin in human plasma using guaifenesin-d3 as internal standard (IS). Following solid phase extraction, the analyte was chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in multiple reaction-monitoring (MRM) mode (positive ion mode). The limit of quantitation for this method was 8?ng/mL and the linear dynamic range was 8–2200?ng/mL.     

Determination of tamsulosin in human aqueous humor and serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.     

High-performance liquid chromatographic analysis of a selective cyclooxygenase-1 inhibitor SC-560 in rat serum: application to pharmacokinetic studies

A method of analysis of SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism. A simple high-performance liquid chromatographic method was developed for simultaneous determination of SC-560 and other products of metabolism in rat serum. Serum (0.1 ml) was precipitated with acetonitrile after addition of the internal standard, testosterone 17-propionate. Separation was achieved on a C₈ column with UV-detection at 240 nm. The calibration curve was linear ranging from 0.02 to 100 μg/ml. The mean recovery was >86.7%. Precision of the assay was <10% (R.S.D.%), and was within 15% at the limit of quantitation (20 ng/ml). Bias of the assay was lower than 15.5%. The limit of detection was 10 ng/ml for a 0.1 ml sample. The assay was applied successfully to the in vivo kinetic study of SC-560 in rats.