A rapid and simple high-performance liquid chromatography method for the determination of human plasma levofloxacin concentration and its application to bioequivalence studies

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of levofloxacin in human plasma is described. Neutralized with phosphate buffer (pH 7.0), the sample (0.1 mL) was extracted with dichlormethane (1 mL). After voltex-mixing and centrifuged at 3000g for 6 min at 4 degrees C, the upper aqueous layer was aspirated using a micro vacuum pump and the organic layer was directly transferred to a clean test tube without pipetting. The organic solvent was evaporated and the residues were reconstituted with the mobile phase. Levofloxacin and terazosin (internal standard, IS) were chromatographically separated on a C(18) column with a mobile phase containing phosphate buffer (pH 3.0, 10 mm), acetonitrile and triethylamine (76:24:0.076, v/v/v) at a flow rate of 1 mL/min. The analytes were detected using fluorescence detection at an excitation and emission wavelength of 295 and 440 nm, respectively. The linear range of the calibration curves was 0.0521-5.213 microg/mL for levofloxacin with a lower limit of quantitation (0.0521 microg/mL). The retention times of levofloxacin and terazosin were 2.5 and 3.1 min, respectively. Within- and between-run precision was less than 12 and 11%, respectively. Accuracy ranged from -6.3 to 4.5%. The recovery ranged from 86 to 89% at the concentrations of 0.0521, 0.5213 and 5.213 microg/mL. The present HPLC-FLD method is sensitive, efficient and reliable. The method described herein has been successfully used for the pharmacokinetic and bioequivalence studies of a levofloxacin formulation product after oral administration to healthy Chinese volunteers.     

A simple method for nicardipine hydrochloride quantification in plasma using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple and sensitive reversed-phase liquid chromatography method was developed and validated for the determination of nicardipine hydrochloride (NC) in rabbit plasma. Nicardipine hydrochloride and nimodipine, used as internal standard, were initially extracted from plasma by a rapid solid-phase extraction using C(18) cartridges. After extraction, nicardipine hydrochloride was separated by HPLC on a C(18) column and quantified by ultraviolet detection at 254 nm. A mixture of acetonitrile-0.02 M sodium phosphate buffer-methanol (45:40:15) with 0.2% of triethylamine of pH of 6.1 was used as mobile phase. The mean (+/-SD) extraction efficiency of NC was 77.56 +/- 5.4, 84.23 +/- 4.32 and 83.94 +/- 3.87% for drug concentrations of 5, 25 and 100 ng/mL, respectively. The method proved to be linear in the range of 5-100 ng/mL with a regression coefficient of 0.9993. The relative standard deviations of intra- and inter-day analysis for NC in plasma were 3.26-6.52% (n = 5) and 4.71-9.38% (n = 5), respectively. The differences of the mean value measured from the concentration prepared, expressed in percentages (bias percentage), were only - 5.2, 0.4 and 0.8% at NC 5, 25 and 50 ng/mL, which confirmed the accuracy of the method. The analytical technique was used to determine NC plasma concentration after drug oral administration to rabbits. The results inferred that NC is rapidly absorbed in rabbits and has a short half-life (t(1/2) = 1.34 h).     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Simultaneous quantification of olanzapine and its metabolite N‐desmethylolanzapine in human plasma by liquid chromatography tandem mass spectrometry for therapeutic drug monitoring

A simple, sensitive, and selective liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of olanzapine (OLZ) and its metabolite N‐desmethylolanzapine (DMO) in human plasma for therapeutic drug monitoring. Sample preparation was performed by one‐step protein precipitation with methanol. The analytes were chromatographed on a reversed‐phase YMC‐ODS‐AQ C₁₈ Column (2.0 × 100 mm,3 µm) by a gradient program at a flow rate of 0.30 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in positive ion mode. The method was validated for selectivity, linearity, accuracy, precision, matrix effect, recovery and stability. The calibration curve was linear over the concentration range 0.2–120 ng/mL for OLZ and 0.5–50 ng/mL for DMO. Intra‐ and interday precisions for OLZ and DMO were <11.29%, and the accuracy ranged from 95.23 to 113.16%. The developed method was subsequently applied to therapeutic drug monitoring for psychiatric patients receiving therapy of OLZ tablets. The method seems to be suitable for therapeutic drug monitoring of patients undergoing therapy with OLZ and might contribute to prediction of the risk of adverse reactions. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug–drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C₁₈ column and quantitated with a 16 min run in a triple‐quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC‐MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

Liquid chromatography/electrospray ionization tandem mass spectrometry for the quantification of mitiglinide in human plasma: validation and its application to pharmacokinetic studies

A sensitive and specific method was developed and validated for the determination of mitiglinide in human plasma using liquid chromatographic separation with electrospray ionization tandem mass spectrometric detection. Acidified plasma samples were extracted with ethyl acetate. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C(18) column with a mobile phase of methanol-10 mm ammonium acetate solution at a flow rate of 0.3 mL/min. Analytes were detected with an Agilent 6410 Triple qudrupole mass spectrometer equipped with an electrospray ionization source in positive multiple reaction monitoring mode: m/z 316.2 (precursor ion) to 298.2 (product ion) for mitiglinide and m/z 318.2 (precursor ion) to 120.2 (product ion) for the internal standard. This method was validated over a linear range of 0.5-4000 ng/mL for mitiglinide in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/mL, while a relative standard deviation (RSD) was less than 3.9%. The intra- and inter-run precision (as RSD, %) obtained from three validation runs were all less than 15%. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

High-performance liquid chromatography and LC-ESI-MS method for the identification and quantification of two biologically active polyisoprenylated benzophenones xanthochymol and isoxanthochymol in different parts of Garcinia indica

A reverse-phase high-performance liquid chromatographic method was developed for qualitative and quantitative analysis of xanthochymol (1), and isoxanthochymol (2) in the fruit rinds, leaves and seed pericarps of Garcinia indica with confirmation using PDA detection and electrospray ionization MS. Absorption at 276 nm was chosen as the measuring wavelength at which resolution and baseline separation of compounds (1) and (2) could be obtained. The identity of the above two isomeric compounds (1) and (2) in the samples was unambiguously determined by their respective quasi-molecular ion [M - H]- in ESI-MS. Compounds (1) and (2) were qualitatively and quantitatively analyzed in the above three samples of Garcinia indica. The overall analytical procedure is rapid and reproducible and is considered for the analysis of the above two compounds.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as 'estoraque do campo' or 'cuia de brejo', has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts.     

Development and validation of a high performance liquid chromatography quantification method of levo‐tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study

Levo ‐tetrahydropalmatine (l‐ THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l‐ THP and its desmethyl metabolites l‐ corydalmine (l‐ CD) and l‐ corypalmine (l‐ CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid–liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed‐phase Symmetry® C₁₈ column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile–methanol–10 mm ammonium phosphate (pH 3) (10:30:60, v /v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1–10,000 ng/mL. The intra‐ and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l‐ THP in rats. Taken together, the developed method can be applied for bioanalysis of l‐ THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.     

Simultaneous determination of ZLR-8 and its active metabolite diclofenac in dog plasma by high-performance liquid chromatography

ZLR-8 is a nitric oxide releasing derivative of diclofenac for the treatment of inflammation. In this paper, a sensitive and reliable high-performance liquid chromatography method for simultaneous determination of ZLR-8 and its active metabolite diclofenac in the plasma of beagle dogs has been developed and validated. After the addition of ketoprofen as the internal standard (IS), plasma samples were extracted with n-hexane-isopropanol (95:5, v/v) mixture solution and separated by HPLC on a reversed-phase C(18) column with a mobile phase of gradient procedure. Analytes were determined by the UV detector which was set at 280 nm. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of ZLR-8 and diclofenac were linear over the range 0.05-4.0 microg/mL. The within- and between-batch precisions (RSD%) were lower than 10% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.05 microg/mL. The proposed method has been readily implemented in preclinical pharmacokinetics studies of ZLR-8 and its active metabolite diclofeance. Representative plasma concentration vs time profiles resulting from administration of ZLR-8 to beagle dogs are presented in this communication.     

Rapid ultraperformance liquid chromatography–tandem mass spectrometry method for quantification of oxcarbazepine and its metabolite in human plasma

A rapid and sensitive ultraperformance liquid chromatography tandem mass spectrometry assay was developed for the simultaneous analysis of oxcarbazepine and its main metabolite in human plasma. The assay involves a simple solid‐phase extraction procedure of 0.3 mL of human plasma and analysis was performed on a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. Separation was achieved on an Acquity UPLC™ BEH C₁₈ column (50 × 2.1 mm, i.d., 1.7 µm) with isocratic elution at a flow‐rate of 0.25 mL/min and imipramine was used as the internal standard. The standard calibration curve was linear over the range 9.580–5070.205 ng/mL for oxcarbazepine (OXC) and 19.444–10290.800 ng/mL for 10,11‐dihydro‐10‐hydroxycarbamazepine (MHD), expressed by the linear correlation coefficient r², which was better than 0.995 for OXC and MHD. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recoveries were 81.0, 89.6 and 66.6% for OXC, MHD and imipramine, respectively. The total run time was 1.5 min only for each sample, which makes it possible to analyze more than 350 samples per day. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.     

A sensitive and selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer

We describe a selective and a highly sensitive assay for actinomycin-D (Act-D) and vincristine (VCR) in plasma employing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection. The intraday precision (as defined by the coefficient of variation, CV) based on the standard deviation of replicates of quality control samples ranged from 4.9 to 7.5% and 6.5 to 11.3% with accuracy ranging from 90.7 to 98.1% and 91.2 to 103% for Act-D and VCR, respectively. The interday precision ranged from 7.2 to 10.0% and 11.3 to 13.0% and the accuracy ranged from 94.3 to 102% and 90.7 to 91.6% for Act-D and VCR, respectively. Stability studies showed that Act-D and VCR were stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) for both Act-D and VCR was 0.05 ng/ml. The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and is being successfully employed in a pharmacokinetic study of these agents in children with cancer, and is expected to support several ongoing and future pediatric trials.     

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital‐pretreated rats and detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3′(R)‐hydroxy‐nodakenetin‐3′‐ol and 3′(S)‐hydroxy‐nodakenetin‐3′‐ol, respectively, on the basis of their ¹H‐NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP‐HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C₁₈ column, a mobile phase with MeOH‐H₂O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat‐inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50–400, 20–120 and 150–24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd.     

A liquid chromatography-mass spectrometry method for the quantification of both etoricoxib and valdecoxib in human plasma

A practicable and selective liquid chromatography-mass spectrometry assay for the determination of two cyclooxygenase-2 inhibitors, etoricoxib and valdecoxib, in human plasma is presented. The analytical technique is based on reversed-phase high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry (Finnigan Mat LCQ ion trap). Mass analysis was performed in the positive ion mode. The ion trap was operated in the tandem MS mode (MS2) and the transitions of etoricoxib (m/z 359.2 --> 280.3) and valdecoxib (m/z 315.1 --> 235.1) were followed by selected reaction monitoring. Retention times of etoricoxib and valdecoxib were 1.05 and 1.08 min, respectively. The method was validated over a linear range 10-2500 and 5-1000 microg/L using the other substrate as internal standard. After validation, the method was used to study the pharmacokinetic pro fi le of etoricoxib or valdecoxib in a healthy volunteer after administration of a single oral dose (valdecoxib, 20 mg; etoricoxib, 90 mg). The presented method was suf fi cient to cover more than 90% of the area under the plasma concentration time curve.     

Comparison of a high‐performance liquid chromatography method for quantification of carbamazepine with chemiluminescent microparticle immunoassay

Carbamazepine is an antiepileptic drug widely used for the treatment of epilepsy. In the National Institute of Neurology, monitoring has been performed using the technique chemiluminescent microparticle immunoassay (CMIA) in an automated way during the last five years. The aim of this study was to develop a simple and rapid HPLC analytical method coupled to DAD‐UV detection for the determination of plasma concentrations of carbamazepine and compare its feasibility with those used in routine analysis. The developed HPLC method was fully validated and the applicability of the proposed method was verified through the analysis of plasma samples of patients and later compared with the quantification of the same plasma samples with the CMIA method. The limit of quantification obtained was 0.5 μg/mL. The mean value for recovery was 99.05% and the coefficient of variation (CV) was 5.6%. The precision and accuracy of this method were within the acceptable limits; inter‐ and intraday CV values were <10%. The correlation between the CMIA method and the developed HPLC method was very good (r ≈ 0.999). A Bland–Altman plot showed no significant bias between the results. The HPLC‐DAD method may be an alternative to determine and monitoring the carbamazepine levels in human plasma or serum. Copyright © 2015 John Wiley & Sons, Ltd.