Quantitation of niflumic acid in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study of talniflumate tablets

A rapid and simple HPLC method with UV detection (288 nm) was developed and validated for quantitation of niflumic acid in human plasma, the active metabolite of talniflumate. After precipitation with 100% methanol containing the internal standard, indomethacin, the analysis of the niflumic acid level in the plasma samples was carried out using a reverse phase C18 CAPCELL PAK (5 microm, 4.6 mm x 250 mm) column. The chromatographic separation was accomplished with an isocratic mobile phase consisting of a mixture of 0.1M sodium acetate in water and acetonitrile (37:63, v/v), adjusted to pH 6.4. This HPLC method was validated by examining its precision and accuracy for inter- and intra-day runs in a linear concentration range of 0.02-5.00 microg/mL. Stability of niflumic acid in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method was successfully applied to the bioequivalence study of talniflunate in healthy volunteers.     

Quantitative determination of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass spectrometry and its application to a bioequivalence study

A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 microl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis HLB column (1 mm x 50 mm, 30 microm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column (4.6 mm x 50 mm, 5 microm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/x2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at -30 degrees C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos formulation (encapsulated 15 mg tablet) and an Actos 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos 15 mg tablet.     

An optimized analytical method of fluconazole in human plasma by high-performance liquid chromatography with ultraviolet detection and its application to a bioequivalence study

A sensitive and accurate HPLC-UV method for the quantification of fluconazole (FLA) level in human plasma has been developed. The sample was prepared by one-step liquid-liquid extraction (LLE) of FLA from plasma using dichloromethane. Phenacetin was used as the internal standard. The chromatographic retention times of FLA and phenacetin were 4.6 and 8.3 min, respectively. The lower limit of quantitation (LLOQ) was 0.05 microg/mL, and no interferences were detected in the chromatograms. The devised HPLC-UV method was validated by evaluating its intra- and inter-day precisions and accuracies in a linear concentration range between 0.05 and 10.00 microg/mL. The devised method was successfully applied to a bioequivalence studies involving the oral administration of a single 150 mg FLA tablet and 3 x 50 mg FLA capsules in healthy Korean male volunteers.     

Quantitation of monohydroxy fatty acids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) procedure for the separation of hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecanoic acids (HODEs) after derivatization of the hydroxy group with 1-anthroylnitrile is described. Anthroyl esters of HETEs were separated from those of HODEs by reversed-phase HPLC. The positional isomers of the HETEs and HODEs were well separated by normal-phase HPLC. The fluorimetric HPLC method has a high sensitivity and naturally occurring HETEs can be quantitatively analyzed at the picomolar level. The amount of 5-HETE in A23187-stimulated polymorphonuclear leukocytes (PMNLs) was determined by the present method. PMNLs produced approximately 150 ng of 5-HETE per 10⁷ cells at 5 min stimulation. The amount of 5-HETE determined by fluorimetric detection was consistent with that determined by ultraviolet detection (235 nm).     

Determination of bestatin in serum by high-performance liquid chromatography with fluorescence detection

A simple method for the determination of bestatin and its major metabolite in man, p-hydroxybestatin, in human serum was investigated; the method employs high-performance liquid chromatography with fluorescence detection. Bestatin and p-hydroxybestatin are oxidized to phenylacetaldehyde and p-hydroxyphenylacetaldehyde, respectively, with periodate, which are then converted into fluorescent compounds with 4,5-dimethoxy-1,2-diaminobenzene. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18. The detection limits of bestatin and p-hydroxybestatin are 0.2 and 0.4 μg/ml serum, respectively. This method permits the precise determination of bestatin in serum (20 μl) from patients administered bestatin. p-Hydroxybestatin in serum can not be measured by this method because of its low concentration (less than the detection limit).     

Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fluorescence detection: its application to a bioequivalence study

A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride-acetonitrile solution in borate buffer (pH=8.0) at 30 degrees C for 30 min. The chromatographic separation was performed on a Diamonsil C18 column (150.0 mmx4.6 mm, 5 microm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1-10.0 microg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were 相似文献   

Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: application to a bioequivalence study

A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L(-1); pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025-10 microg mL(-1) of clarithromycin in human serum with a limit of quantification of 0.025 microg mL(-1). The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.     

Determination of centpropazine and its metabolite in rat serum by high-performance liquid chromatography and fluorescence detection

A new HPLC assay method was developed for the simultaneous assay for centpropazine (antidepressant) and its hydroxylated metabolite (II) to assess their pharmacokinetics and metabolism characteristics. Rat serum samples were extracted with ether, backwashed with n-hexane and injected onto the HPLC system, which used a C₁₈ column, gradient elution and fluorescence detection at 250 Ex/350 nm Em. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of <±20% at low and <±15% at higher concentrations. Samples were stable in autosampler prior to injection and after multiple freeze–thaw cycles. Linearity was observed between 0.625 and 20 ng/ml for both I and II in serum. Overall the method developed was highly sensitive and could be employed for a wide range of studies.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Determination of polyamines in human saliva by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250×4.6 mm I.D.; 5 μm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.     

Quantitation of harringtonine and homoharringtonine in serum by high-performance liquid chromatography

Harringtonine and homoharringtonine are naturally occurring alkaloids with demonstrated antineoplastic activity against certain types of leukemias in cell cultures, experimental animals, and initial clinical trials. Sample preparation consists of addition of the internal standard (one compound used as the internal standard for the other), solvent extraction with methylene chloride, washing with ammonium formate, and evaporation to dryness. The residue is dissolved in the mobile phase (40% methanol—60% 0.1M ammonium formate) and an aliquot is chromatographed on μC₁₈ reversed-phase column (flow-rate 1.5 ml/min). Peaks are detected with a spectrophotofluorimeter by monitoring the emission at 320 nm with excitation wavelength of 280 nm. Limit of detection is 10 ng/ml (20 nM) for both compounds; reproducible quantitation can be made to 30 ng/ml (60 nM).