Simultaneous determination of oxycodone and its major metabolite, noroxycodone, in human plasma by high-performance liquid chromatography

Oxycodone (14-hydroxy-7,8-dihydrocodeinone) is a potent opioid receptor agonist. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of oxycodone and its major metabolite, noroxycodone, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile and phosphate buffer (8:92, v/v) at a flow rate of 1 mL/min, and UV detection at 205 nm. The retention times for oxycodone, noroxycodone and codein (internal standard) were 14.7, 13.8 and 10.2 min, respectively. The validated quantitation range of the method was 2-100 ng/mL for oxycodone and 10-100 ng/mL for noroxycodone. The developed procedure was applied to assess the pharmacokinetics of oxycodone and its metabolite following administration of a single 20 mg oral dose of oxycodone hydrochloride to one healthy male volunteer.     

The use of liquid chromatography/mass spectrometry for quantitative analysis of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were <11.3% and <+/-14.9%, respectively, and the inter-day precision and accuracy were <14.9% and <+/-6.5%, respectively, for all the concentrations and matrices described.     

Determination of artemether in plasma and whole blood using HPLC with flow-through polarographic detection

An HPLC method with polarographic detection for the trace determination of artemether in plasma and whole blood was developed and applied to pharmacokinetic and clinical pharmacological studies. The method showed high sensitivity and selectivity because of the easy reduction of the peroxide linkage of artemether at the mercury drop electrode. The detection limit was 10 ng and the detector response was linear over the range of 10 ng to 1 microgram artemether injected onto the column. The largest relative standard deviation of 10 replicate measurements of standard solutions (concentrations of 10 ng/mL-1 microgram/mL) was 8%. The recovery from whole blood and plasma of added drug (concentrations of 15-480 ng/mL) was 71-100%.     

Determination of oxycodone and hydrocotarnine in cancer patient serum by high-performance liquid chromatography with electrochemical detection.

We developed an HPLC procedure using electrochemical detection for the quantitation of oxycodone and hydrocotarnine in cancer patients serum. An eluent of methanol:acetonitrile:5 mM pH 8 phosphate buffer (2:1:7) was used for the mobile phase. The calibration curve was linear in the range from 10 ng/mL to 100 ng/mL. The recovery of oxycodone and hydrocotarnine was 97.2% and 90.5%, respectively. The relative standard deviations within-runs and between-runs for the assay of oxycodone or hydrocotarnine were less than 4.8%. The method developed here was better than the method reported previously.     

Development and validation of a reversed-phase HPLC method for determination of nitrendipine in rat plasma: application to pharmacokinetic studies

A simple and sensitive method for the determination of nitrendipine in rat plasma was developed using high-performance liquid chromatography (HPLC). The procedure involves extraction of nitrendipine in dichloromethane/sodium hydroxide, followed by reversed phase HPLC using a Waters, Spherisorb ODS2 (250 x 4.6 mm, 5 microm) column and UV detection at 238 nm. The retention times of nitrendipine and internal standard (felodipine) were 5.0 min and 7.5 min, respectively. The calibration curves were linear over the range of 5 ng/mL (lower limit of quantification, LOQ) to 200 ng/mL for nitrendipine. The intra- and inter-day coefficients of variation for all criteria of validation were less than 15% over the linearity range. The sensitivity and precision of the method were within the accepted limits (< 15%) throughout the validation period. The present method was also successfully applied for the study of plasma pharmacokinetics of nitrendipine loaded solid lipid nanoparticles (SLN) in rats.     

High-performance liquid chromatography-electrospray ionization-mass spectrometric determination of emedastine difumarate in human plasma and its pharmacokinetics

A selective and sensitive method employing high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry is developed and validated for the determination of emedastine difumarate in human plasma. With naphazoline hydrochloride as the internal standard, emedastine difumarate is extracted from plasma with ethyl acetate. The organic layer is evaporated, and the residue is redissolved in the mobile phase. An aliquot of 10 microL is chromatographically analyzed on a prepacked Phenomenex Luna 5u CN 100A (150 x 2.0-mm i.d.) column, using a mobile phase comprised of methanol-water (20 mM CH(3)COONH(4), pH 4.0) (80:20, v/v). Standard curves are linear (r(2) = 0.9990) over the concentration range of 0.05-30 ng/mL and had good accuracy and precision. The within- and between-batch precisions did not exceed 15% for the relative standard deviation. The lower limit of detection is 0.01 ng/mL. The validated HPLC-ESI-MS method is successfully used to study emedastine difumarate pharmacokinetics in 12 healthy volunteers.     

Assay of ibuprofen in human plasma by rapid and sensitive reversed-phase high-performance liquid chromatography:application to a single dose pharmacokinetic study

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method is developed to determine the concentration of ibuprofen in human plasma. Ibuprofen is isolated from plasma by adding 0.50 mL of acetonitrile to 1.0 mL of plasma. The endogenous substances precipitated by acetonitrile are separated by centrifugation. The supernatant is saturated with ammonium sulfate to salt-out the acetonitrile. The salted-out acetonitrile is injected directly into the HPLC system. A 150-mm x 4.6-mm column packed with 3-microns reversed-phase octadecylsilane particles (C18) is used for the method finally developed. The mobile phase is a 1:1 ratio of acetonitrile-phosphoric acid (pH 2.2). Ibuprofen is monitored with a UV-visible detector at 220 nm and 0.10-0.002 absorbance units full scale (A.U.F.S.). The mean percent of relative standard deviations for within-day and between-day analyses are less than 3. The limit of detection for ibuprofen (in human plasma) is 25 ng/mL for a 100-microL injection volume. Quantitation of ibuprofen in human plasma at 100 ng/mL can be achieved with a relative standard deviation of less than 5%. The completion time of assay is less than 20 minutes.     

Nanogram level quantitation of oxycodone in human plasma by capillary gas chromatography using nitrogen-phosphorus selective detection.

A sensitive and specific capillary gas chromatographic assay is reported for the quantitation of oxycodone in human plasma. The technique involves a single extraction of oxycodone and internal standard (hydrocodone) from plasma by toluene containing 1% isopropanol. Separation is achieved on a methyl silicone (HP-1) fused-silica capillary column (25 m x 0.2 mm I.D., 0.33 microns film thickness) and detection is by nitrogen-phosphorus selective mode. The minimum quantifiable limit is 1.8 ng/ml using 2 ml of plasma. The method is applicable to characterize the plasma profile of oxycodone in humans after a single oral 5-mg oxycodone hydrochloride tablet.     

An HPLC method with diode array detector for the simultaneous quantification of chloroquine and desethylchloroquine in plasma and whole blood samples from Plasmodium vivax patients in Vietnam,using quinine as an internal standard

A sensitive, simple method for quantification of chloroquine (CQ) and desethylchloroquine (MCQ) in whole blood and plasma from Plasmodium vivax patients has been developed using HPLC with diode array detection (DAD). Solid‐phase extraction on Isolute‐96‐CBA was employed to process 100 μL of plasma/whole blood samples. CQ, MCQ and quinine were separated using a mobile phase of phosphate buffer 25 mm , pH 2.60–acetonitrile (88:12, v/v) with 2 mm sodium perchlorate on a Zorbax SB‐CN 150 × 4.6 mm, 5 μm column at a flow rate of 1.2 mL/min, at ambient temperature in 10 min, with the DAD wavelength of 343 nm. The method was linear over the range of 10–5000 ng/mL for both CQ and MCQ in plasma and whole blood. The limit of detection was 4 ng/mL and limit of quantification was 10 ng/mL in both plasma and blood for CQ and MCQ. The intra‐, inter‐ and total assay precision were <10% for CQ and MCQ in plasma and whole blood. In plasma, the accuracies varied between 101 and 103%, whereas in whole blood, the accuracies ranged from 97.0 to 102% for CQ and MCQ. The method is an ideal technique with simple facilities and instruments, bringing about good separation in comparison with previous methods. © 2016 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

Improved HPLC method for doxorubicin quantification in rat plasma to study the pharmacokinetics of micelle-encapsulated and liposome-encapsulated doxorubicin formulations

An improved simple, rapid and accurate HPLC method for quantification of doxorubicin derived from micelle-encapsulated or liposome-encapsulated doxorubicin formulation in rat plasma was described. The mobile phase consisting of a mixture of methanol-water [containing 0.1% formic acid anhydrous and 0.1% ammonia solution (25%), pH 3.0], 60:40, was delivered at a flow rate of 1.0 mL/min. Sample preparation for micelle- or liposome-encapsulated doxorubicin in rat plasma were achieved directly by protein precipitation with acetonitrile. Doxorubicin and daunorubicin (internal standard, IS) were separated on a C(18) reversed-phase HPLC column and quantified by a fluoresence detection with an excitation wavelength of 475 nm and an emission wavelength of 580 nm. The linearity was obtained over the range of 5.0-1000.0 ng/mL and 1.0-200.0 microg/mL for doxorubicin and the lower limit of quantitation was 5.0 ng/mL. For each level of quality control samples, inter- and intra-assay precision was less than 9.6 and 5.1% (relative standard deviation), respectively, and percentage error was within +/-2.6%. The extraction recoveries of doxorubicin in the range of 10 ng/mL to 100 microg/mL in rat plasma were between 94.1 and 105.6%. This method was successfully applied to the pharmacokinetic study of doxorubicin formulations after i.v. administration to rats.     

毛细管电泳-电致化学发光法测定兔血浆中的羟考酮

基于稀土Eu(Ⅲ)掺杂的类普鲁士蓝膜修饰的铂电极为工作电极,建立了测定羟考酮的毛细管电泳-电致化学发光分析方法。考察了检测电位、运行缓冲溶液的酸度及浓度、分离电压、进样条件等对电泳分离效果及检测灵敏度的影响。在最佳的实验条件下,羟考酮可在4 min内得到分离,其ECL强度值与羟考酮的质量浓度在7.0×10-2～7.0μg/mL和7.0～70.0μg/mL范围内呈良好的线性关系,检出限为4.2×10-2μg/mL(3σ),峰高和迁移时间的相对偏差分别为3.6%和0.48%(n=6)。方法用于兔血浆中羟考酮含量的检测,加标回收率在99.7%～101.0%之间。     

Quantitative determination of imatinib in human plasma with high-performance liquid chromatography and ultraviolet detection

A simple and sensitive high-performance liquid chromatography (HPLC) method was developed to quantitate imatinib in human plasma. Imatinib and the internal standard dasatinib were separated using a mobile phase of 0.5% KH(2)PO(4) (pH3.5)-acetonitrile-methanol (55:25:20, v/v/v) on a CAPCELL PAK C18 MG II column (250 mm × 4.6 mm) at a flow rate of 0.5 mL/min and measurement at UV 265 nm. Analysis required 100 μL of plasma and involved a solid phase extraction with an Oasis HLB cartridge, which gave recoveries of imatinib from 73% to 76%. The lower limit of quantification for imatinib was 10 ng/mL. The linear range of this assay was between 10 and 5000 ng/mL (regression line r(2) > 0.9992). Inter- and intra-day coefficients of variation were less than 11.9% and accuracies were within 8.3% over the linear range. The plasma concentrations of imatinib obtained by our present method were almost the same as those assayed by an LC-MS-MS method at the Toray Research Center, Inc. This method can be applied effectively to measure imatinib concentrations in clinical samples.     

Usefulness of the chaotropic effect in sample preparation for chromatographic analysis of acidic xenobiotics in human plasma

In this study a new RP‐HPLC with photo‐diode array detection method for the determination of ibuprofen ((RS)‐2‐(4‐isobutylphenyl)propionic acid) in human plasma samples was developed. Samples were prepared by SPE and analyzed by an isocratic elution mode over a C₁₈ column using 80% methanol. A novel sample pretreatment method, based on the addition of ionic liquids possessing chaotropic ions to small human plasma sample (100 μL), was elaborated. 1‐Butyl‐3‐methylimidazolium chloride and 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMIM BF₄) were tested from the point of view of extraction yield. Quantification was based on calibration curve applying diclofenac as the internal standard. Owing to dilution of plasma sample by 2 mM aqueous solution of BMIM BF₄ before SPE, appropriate sample purification and extraction yields higher than 95% with precision lower than 2% can be achieved. Linear coefficients of correlation (r²) were >0.99 in the range of 0.3–5 μg/mL ibuprofen concentration in plasma. The limit of quantification was 65 ng/mL and the detection limit for ibuprofen was 19.5 ng/mL.     

Development and validation of a high‐performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies

A sensitive and selective RP‐HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD‐20A UV–vis) at a λ_max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol–acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100–2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method.     

High-performance liquid chromatographic assay of indomethacin in porcine plasma with applicability to human levels

A high-performance liquid chromatography (HPLC) assay is described for the determination of indomethacin in porcine plasma using acetonitrile to precipitate plasma proteins and for the one-step extraction. Calibration curves (using the internal standard method) are linear (r2 > 0.98) over the concentration range of 50.0 to 3000 ng/mL in both mobile phase and plasma. Precision, expressed as the inter- and intraday coefficient of variation (n = 5), is < 7% on the same day and < 5% between days at each plasma control sample of 300, 1000, and 3000 ng/mL, respectively. System precision, calculated as the coefficient of variation (n = 5), is < 7% at 3000 ng/mL of indomethacin, and the limit of quantitation in plasma is 50 ng/mL. The absolute recovery for both indomethacin and the internal standard (mefenamic acid) from plasma is over 97% (n = 3), and the concentrations do not deviate more than -2.9% to 2.4% from their actual values. The specificity of the method is confirmed. This technique is thus reported to be both rapid and specific. The real advantage is the small sample volume required (500 microL), which allows it to be considered for the quantitation of indomethacin in plasma from paediatric patients.     

High-performance liquid chromatographic method for simultaneous determination of sophoridine and matrine in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of sophoridine and matrine in rat plasma. Sophoridine and matrine in the resulting supernatant of the plasma deproteinized with acetonitrile containing an internal standard (acetanilide) were directly determined by reversed-phase HPLC and ultraviolet detection. The result of limits of quantitation for matrine and sophoridine were 200 and 350 ng/mL in plasma, respectively, and recovery of both analytes was greater than 98%. The assay was linear from 250 to 4000 ng/mL for matrine and from 500 to 8000 ng/mL for sophoridine. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of matrine and sophoridine in the plasma following oral administration of Kexieling tablets, which is one of the preparations of Kudouzi at a dose equivalent to 30 and 60 mg/kg of matrine and sophoridine, respectively.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

An automated analyzer for vancomycin in plasma samples by column-switching high-performance liquid chromatography with UV detection

An automated analyzer for vancomycin in rat plasma by column-switching high-performance liquid chromatography (HPLC) with UV detection was developed. The method includes in-line extraction of vancomycin by ion-exchange cartridge column and a separation on a reversed-phase column with UV detection at 215 nm. Plasma samples were diluted by mobile phase solution and directly injected to HPLC. Vancomycin was quantitatively recovered from rat plasma samples. The separation was completed within 15 min. The calibration curve was linear over the range from 0.5 to 100 microg/mL with the detection and quantification limits of 0.5 microg/mL (2.5 ng on column; signal-to-noise ratio = 3). The values of precision in intra- and inter-day assays (n = 3) were less than 1.92 and 3.69%, respectively. This method does not require time-consuming pre-treatment and is suitable for the routine assay of plasma samples.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.